1 SUPPLEMENTARY FIGURES 2 3 FINE-SCALE ADAPTATIONS TO ENVIRONMENTAL VARIATION AND GROWTH 4 STRATEGIES DRIVE PHYLLOSPHERE DIVERSITY. 5 6 Jean-Baptiste Leducq1,2,3, Émilie Seyer-Lamontagne1, Domitille Condrain-Morel2, Geneviève 7 Bourret2, David Sneddon3, James A. Foster3, Christopher J. Marx3, Jack M. Sullivan3, B. Jesse 8 Shapiro1,4 & Steven W. Kembel2 9 10 1 - Université de Montréal 11 2 - Université du Québec à Montréal 12 3 – University of Idaho 13 4 – McGill University 14

1 15 Figure S1 - Validation of Methylobacterium group A subdivisions in nine clades. Distribution 16 of pairwise nucleotide similarity (PS) within and among Methylobacterium clades and 17 (outgroup) estimated from the complete rpoB nucleotide sequence (4064 bp). PS was 18 calculated in MEGA7 (1, 2) from aligned nucleotide sequences as PS = 1-pdistance. 19

2 A1

A2

A3

A4

A5

A6 Within clades

A9

B

C Within Methylobacterium

among A1−A9

between A and B

between A and C

between B and C

within Microvirga between Methylobacterium and Microvirga

0.75 0.80 0.85 0.90 0.95 1.00

PS 20 Figure S2 - ASV rarefaction on diversity assessed by 16s barcoding in 46 phyllosphere 21 samples, 1 positive control (METH community) and 1 negative controls. a) Diversity in 22 METH community (positive control) and rare ASVs threshold definition. The METH community 23 consisted in mixed genomic DNAs from 18 Methylobacterium isolates representative of diversity 24 isolated in SBL and MSH in 2017 and 2018 (Table S6; Table S10), one Escherichia coli strain 25 and one Sphingomonas sp. isolate from MSH in 2017 (isolate DNA022; Table S6), also probably 26 contaminated with Thermotoga sp. (Table S4). b) Rarefaction curves (Black = phyllosphere 27 samples; red= METH community; blue= negative control). c) Cumulated ASVs relative 28 abundance per sample before rarefaction, from the most abundant to the rarest ASV. d) 29 Cumulated ASVs relative abundance per sample after rarefaction, from the most abundant to the 30 rarest ASV (negative control excluded). ASV definition and rarefaction were conducted in R with 31 package dada2 (3). 32

3

33 Figure S3 - Main order distribution across 46 phyllosphere samples and one 34 positive control (METH community) in function of different factors. For each factor and each 35 bacterial order (color code on top left) ASV relative sequence abundances are shown. 36

4

37 Figure S4 - Community analysis on bacterial diversity assessed from 16S barcoding in 46 38 phyllosphere samples. (a) Principal component analysis (Bray-Curtis distance on ASV relative 39 abundance, Hellinger transformation) and main taxa contribution to axis (color codes in Figure 40 S3). Each sample is represented by a pie chart with taxa relative abundance. Samples cluster 41 according to forest of origin (SBL, MSH; black lines), time of sampling (ellipsoids, dotted lines) 42 and host tree species (grey ellipsoids). b) Summary of PERMANOVA (10,000 permutations on 43 ASV relative abundance, Hellinger transformation) analysis of variance in community. The 44 Venn diagram represent relative contributions of tested factors and their interaction: forest of 45 origin, time of sampling and host tree species. Significance of factors and interactions: “***”: 46 p<0.00l; “**”: p<0.01; “*”: p<0.05. 47

5

48 Figure S5 - ML phylogenetic trees from sucA (a) and rpoB (b) concatenated hypervariable 49 (HV) regions. Trees were drawn from sequences obtained for 20 representative isolates from 50 2017 pilot survey (black circles) and reference genomes. Trees with the highest log likelihood are 51 shown. Bootstraps: only values for node supported by at least 50% of replicated trees are 52 displayed. Phylogenetic tree was rooted on Microvirga and Enterovirga outgroups (Compressed). 53 a) The sucA ML tree was inferred from 3 aligned concatenated HV regions (1,663 bp) available 54 for 189 reference genomes and 14 tested isolates. b) The rpoB ML tree was inferred from 2 55 aligned and concatenated HV regions (1,244 bp) available for 163 reference genomes and the 20 56 tested isolates. c) Consensus clade tree from sucA and rpoB ML phylogenies. Only tree topology 57 among clades supported by both phylogenies is shown, regardless bootstrap support. For each 58 consensus node, the minimum (most conservative) bootstrap support found between phylogenies 59 is shown (grey scale, legend on top). 60

6 100 95 M01-DNA014 M06-LYS051 M01-LYS083 M06-DNA024 M01-DNA006 NZ PDHT01000001.1 Methylobacterium sp. M01-DNA010 M06-DNA006 M06-LYS083 10074 M01-LYS051 NZ LMNU01000001.1:191921-193610 Methylobacterium sp. Leaf125 contig 1 whole genome shotgun sequence 100 M06-DNA014 a. M01-LYS027 b. M06-DNA010 c. Minimum 94 100 NZ PDHT01000004.1:206700-208389 Methylobacterium sp. V23 contig 4 whole genome shotgun sequence 95 M06-DNA001 55 NZ LMMK01000045.1:446445-448134 Methylobacterium sp. Leaf88 contig 5 whole genome shotgun sequence NZ LMNU01000001.1 Methylobacterium sp. NZ LMRA01000034.1:22889-24578 Methylobacterium sp. Leaf465 contig 4 whole genome shotgun sequence 78 M06-LYS027 100 NZ LMRM01000034.1:24853-26542 Methylobacterium sp. Leaf94 contig 4 whole genome shotgun sequence 91 NZ LMRM01000001.1 Methylobacterium sp. 98 NZ LMNE01000045.1:11316-13005 Methylobacterium sp. Leaf111 contig 5 whole genome shotgun sequence NZ LMNE01000001.1 Methylobacterium sp. 67 NZ LMML01000028.1:23646-25335 Methylobacterium sp. Leaf89 contig 5 whole genome shotgun sequence NZ LMRA01000001.1 Methylobacterium sp. node support 90 100 NZ QAYK01000006.1:21974-23663 Methylobacterium sp. GV104 Ga0189710 106 whole genome shotgun sequence NZ LMMK01000001.1 Methylobacterium sp. 100 Clade A1 81 65 NZ LMML01000001.1 Methylobacterium sp. 100 NZ QJTP01000006.1:21974-23663 Methylobacterium sp. GV094 Ga0189709 106 whole genome shotgun sequence 95 Clade A1 NZ LMMZ01000012.1:271219-272908 Methylobacterium sp. Leaf104 contig 2 whole genome shotgun sequence NZ LMMZ01000001.1 Methylobacterium sp. NZ LMNL01000034.1:566043-567732 Methylobacterium sp. Leaf117 contig 4 whole genome shotgun sequence NZ QJTP01000001.1 Methylobacterium sp. NZ QAYK01000001.1 Methylobacterium sp. 100 NZ LMNJ01000007.1:23885-25574 Methylobacterium sp. Leaf113 contig 15 whole genome shotgun sequence 100 0.00 NZ JAAHTB010000001.1:44054-45741 Methylobacterium sp. BTF04 contig00001 whole genome shotgun sequence 79 NZ LMNJ01000001.1 Methylobacterium sp. NZ FOPM01000025.1:26319-28002 Methylobacterium gossipiicola strain Gh-105 whole genome shotgun sequence 100 NZ LMNL01000001.1 Methylobacterium sp. 62 M01-DNA018 NZ FOPM01000077.1 Methylobacterium gossipiicola 86 100 100 NZ VRUZ01000100.1 Methylobacterium sp. NZ VRUZ01000075.1:13597-15286 Methylobacterium sp. WL69 NODE 75 length 22240 cov 15.3948 WL69 spades whole genome shotgun sequence 100 100 NZ LMMU01000001.1:2145302-2146991 Methylobacterium sp. Leaf99 contig 1 whole genome shotgun sequence NZ LMMU01000001.1 Methylobacterium sp. 0.25 92 NZ LMNG01000034.1:48585-50274 Methylobacterium sp. Leaf112 contig 9 whole genome shotgun sequence 90 M06-DNA018 69 Clades 52 NZ LMRE01000034.1:286188-287877 Methylobacterium sp. Leaf469 contig 4 whole genome shotgun sequence NZ LMMW01000001.1 Methylobacterium sp. 80 59 NZ LMMX01000023.1:284519-286208 Methylobacterium sp. Leaf102 contig 3 whole genome shotgun sequence NZ LMNG01000001.1 Methylobacterium sp. 100 NZ LMMI01000001.1 Methylobacterium sp. 100 NZ LMMI01000045.1:74751-76440 Methylobacterium sp. Leaf87 contig 5 whole genome shotgun sequence 100 81 NZ LMRE01000001.1 Methylobacterium sp. 0.50 84 NZ LMMW01000005.1:48140-49829 Methylobacterium sp. Leaf100 contig 13 whole genome shotgun sequence 100 NZ LMME01000008.1:29365-31054 Methylobacterium sp. Leaf85 contig 16 whole genome shotgun sequence 57 NZ LMMX01000001.1 Methylobacterium sp. 100 NZ LMND01000001.1 Methylobacterium sp. 100 NZ LMNB01000034.1:29401-31090 Methylobacterium sp. Leaf106 contig 7 whole genome shotgun sequence NZ AQVT01000001.1:4441637-4443326 Methylobacterium sp. 88A A3OMDRAFT contig1.1 C whole genome shotgun sequence NZ LMRC01000001.1 Methylobacterium sp. Clade A3 NZ VRVC01000082.1:21135-22824 Methylobacterium sp. WL19 NODE 82 length 23583 cov 13.6008 WL19 spades whole genome shotgun sequence 100 NZ LMQK01000001.1 Methylobacterium sp. 100 0.75 NZ KI912577.1:1190301-1191990 Methylobacterium sp. 10 K368DRAFT scaffold00001.1 whole genome shotgun sequence NZ LMMP01000001.1 Methylobacterium sp. 99 M01-LYS093 Clade A2 NZ LMMG01000001.1 Methylobacterium sp. A1 NZ LMMS01000007.1:29396-31085 Methylobacterium sp. Leaf93 contig 15 whole genome shotgun sequence NZ KB910516.1 Methylobacterium sp. 100 97 NZ LMMP01000009.1:69817-71506 Methylobacterium sp. Leaf91 contig 17 whole genome shotgun sequence M06-LYS093 100 NZ LMMG01000041.1:169880-171569 Methylobacterium sp. Leaf86 contig 6 whole genome shotgun sequence 100 M06-LYS069 1.00 69 100 NZ KB910516.1:3909206-3910895 Methylobacterium sp. 77 scaffold1 whole genome shotgun sequence NZ KI912577.1 Methylobacterium sp. 57 Clade A2 NZ LMND01000001.1:716853-718542 Methylobacterium sp. Leaf108 contig 1 whole genome shotgun sequence NZ VRVC01000100.1 Methylobacterium sp. NZ LMQK01000001.1:864145-865834 Methylobacterium sp. Leaf399 contig 1 whole genome shotgun sequence Clade A3 NZ LMMS01000001.1 Methylobacterium sp. 100 NZ LMRC01000032.1:163224-164913 Methylobacterium sp. Leaf466 contig 5 whole genome shotgun sequence NZ LMNB01000001.1 Methylobacterium sp. 100 87 NZ LMME01000001.1 Methylobacterium sp. NZ VRVE01000002.1:26309-27998 Methylobacterium sp. WL9 NODE 2 length 178199 cov 16.6122 WL9 spades whole genome shotgun sequence 66 100 NZ BJZT01000004.1:89218-90907 Methylobacterium haplocladii strain NBRC 107714 sequence004 whole genome shotgun sequence NZ AQVT01000001.1 Methylobacterium sp. NZ JACIDN010000002.1:271977-273666 Methylobacterium brachythecii strain DSM 24105 Ga0373233 02 whole genome shotgun sequence Clade A4 72 NZ VRVB01000100.1 Methylobacterium sp. 99 NZ VRUY01000100.1 Methylobacterium sp. 72 NZ BJZV01000001.1:389127-390812 Methylobacterium gnaphalii strain NBRC 107716 sequence001 whole genome shotgun sequence 84 NZ JACHOP010000002.1:48421-50110 rhodinum strain DSM 2163 Ga0373200 02 whole genome shotgun sequence NZ VRUU01000100.1 Methylobacterium sp. 83 53 NZ LMQV01000043.1:24547-26232 Methylobacterium sp. Leaf456 contig 8 whole genome shotgun sequence NZ VRUV01001000.1 Methylobacterium sp. 98 100 A2 NZ FOSV01000014.1:39102-40791 Methylorubrum salsuginis strain CGMCC 1.6474 whole genome shotgun sequence M06-LYS037 M06-DNA007 86 NZ SCOS01000036.1:11032-12717 Methylobacterium sp. L1A1 NODE 36 length 49412 cov 18.238301 whole genome shotgun sequence 100 Clade A6 NZ QJJJ01000002.1:134129-135818 Methylobacterium sp. B4 Ga0157232 102 whole genome shotgun sequence NZ VRVH01000100.1 Methylobacterium sp. 100 NZ CP047608.1:1641106-1642795 Methylobacterium sp. CLZ complete genome M06-DNA013 100 96 NZ VRUX01001000.1 Methylobacterium sp. NZ CP047607.1:1641118-1642807 Methylobacterium sp. NI91 chromosome complete genome NZ VRUW01000100.1 Methylobacterium sp. 51 NZ CP029173.1:2017332-2019021 Methylobacterium sp. DM1 chromosome complete genome 82 99 NZ VRVF01000100.1 Methylobacterium sp. NZ RCIG01000014.1:91705-93394 Methylobacterium sp. DB1607 whole genome shotgun sequence 100 NZ VRUT01000100.1 Methylobacterium sp. NZ KB316283.1:412612-414301 Methylobacterium sp. MB200 Scaffold2 whole genome shotgun sequence 96 100 NZ KB911270.1 Methylobacterium sp. NZ JACHWM010000002.1:250415-252104 Methylobacterium sp. R2-1 Ga0299752 02 whole genome shotgun sequence 98 NZ FNVI01000084.1 Methylobacterium sp. NZ AP014809.1:1827385-1829074 Methylorubrum populi strain P-1M 73 62 75 NZ BADE01001622.1 Methylobacterium sp. NZ JACHOS010000008.1:250539-252228 Methylorubrum rhodesianum strain DSM 5687 Ga0373205 08 whole genome shotgun sequence 100 66 NZ SLXZ01000001.1 Methylobacterium sp. NZ CP039546.1:1687704-1689393 Methylorubrum populi strain YC-XJ1 chromosome complete genome NZ FOCS01000079.1 Methylobacterium sp. 78 NZ WEKV01000008.1:286965-288654 Methylorubrum populi strain Pinkel Pinkel 08 whole genome shotgun sequence 75 100 NZ FNWP01000067.1 Methylobacterium sp. A3 NZ JACJIB010000001.1:59347-61036 Methylorubrum thiocyanatum strain DSM 11490 Ga0415277 01 whole genome shotgun sequence 100 NZ LMPY01000001.1 Methylobacterium sp. NZ LSNC01000048.1:29536-31225 Methylorubrum populi strain CD11 7 CD11 7 contig 30 consensus whole genome shotgun sequence Clade B 50 86 NZ FOMM01000067.1 Methylobacterium sp. 100 NC 010725.1:1709595-1711284 Methylorubrum populi BJ001 complete sequence 65 100 100 NZ FOZY01000099.1 Methylobacterium sp. 74 NZ SRHQ01000002.1:322076-323765 Methylorubrum sp. Q1 whole genome shotgun sequence NZ CP017640.1 Methylobacterium sp. NZ CP006992.1:1634904-1636593 Methylobacterium sp. AMS5 chromosome complete genome NZ LACA01000001.1 Methylobacterium radiotolerans NZ LMNS01000039.1:212055-213744 Methylobacterium sp. Leaf123 contig 8 whole genome shotgun sequence 65 NC 010505.1 Methylobacterium radiotolerans NC 012988.1:2207362-2209051 AM1 complete genome 92 NZ BJXP01000001.1 Methylobacterium radiotolerans NZ CP019322.1:5387833-5389516 Methylorubrum extorquens strain PSBB040 chromosome complete genome 79 NZ NKQS01000001.1 Methylobacterium radiotolerans 100 NZ AGJK01000053.1:18763-20452 Methylorubrum extorquens DSM 13060 ctg1129 whole genome shotgun sequence NZ QEKZ01000001.1 Methylobacterium organophilum NC 012808.1:1609596-1611285 Methylorubrum extorquens AM1 complete sequence 100100 NZ SAUO01000001.1 Methylobacterium radiotolerans NZ CP021054.1:3391241-3392930 Methylorubrum zatmanii strain PSBB041 chromosome complete genome NZ ANPA01000029.1 Methylobacterium mesophilicum 67 NZ LT962688.1:4087617-4089306 Methylorubrum extorquens strain TK 0001 chromosome TK0001 78 NZ FNHS01000039.1 Methylobacterium phyllostachyos NC 011757.1:1992002-1993691 Methylorubrum extorquens CM4 complete sequence 73 NZ FMWU01000076.1 Methylobacterium sp. A4 NZ MNAO01000056.1:14218-15907 Methylorubrum extorquens strain CP3 NODE 56 length 18697 cov 17.1841 whole genome shotgun sequence 98 NZ NPKS01000094.1 Methylobacterium sp. NZ BJVP01000002.1:284480-286169 Methylorubrum extorquens strain NBRC 15911 sequence02 whole genome shotgun sequence 100 NZ CP003811.1 NC 010172.1:1837249-1838938 Methylorubrum extorquens PA1 complete sequence NZ CP016429.1 Methylobacterium sp. Clade A9 NZ JACJIN010000007.1:302783-304472 Methylobacterium sp. RAS18 Ga0372453 07 whole genome shotgun sequence 97 NZ BACT01001090.1 Methylobacterium sp. 99 NZ CP029550.1:2708178-2709867 Methylobacterium durans strain 17SD2-17 chromosome complete genome 98 99 99 NZ JQJG01000001.1 Methylobacterium sp. NZ SMNT01000065.1:26486-28175 Methylobacterium segetis strain 17J42-1 scf7180000000982 whole genome shotgun sequence 89 95 NZ FPBW01000080.1 Methylobacterium sp. NZ BJZU01000085.1:880-2569 Methylobacterium oxalidis strain NBRC 107715 sequence085 whole genome shotgun sequence Clade A5 NZ CP015367.1 Methylobacterium phyllosphaerae NZ VZZJ01000011.1:156130-157819 Methylobacterium sp. YIM 132548 Scaffold11 whole genome shotgun sequence 89 100 NZ FOPK01000079.1 Methylobacterium phyllosphaerae 100 NZ VZZK01000070.1:12447-14136 strain YIM 48816 Scaffold70 whole genome shotgun sequence 52 NZ AKFK01000001.1 Methylobacterium sp. NZ VRVB01000061.1:18342-20031 Methylobacterium sp. WL30 NODE 61 length 28399 cov 5.3305 WL30 spades whole genome shotgun sequence NZ FOQW01000048.1 Methylobacterium brachiatum 66 70 NZ VRUV01000120.1:1287-2976 Methylobacterium sp. WL116 NODE 120 length 10440 cov 17.1519 WL116 spades whole genome shotgun sequence NZ CP033231.1 Methylobacterium brachiatum NZ VRUY01000143.1:1298-2987 Methylobacterium sp. WL93 NODE 143 length 11205 cov 17.2565 WL93 spades whole genome shotgun sequence NZ LHCD01000001.1 Methylobacterium sp. 100 NZ VRUU01000089.1:8410-10099 Methylobacterium sp. WL119 NODE 89 length 19229 cov 14.5878 WL119 spades whole genome shotgun sequence 95 NZ FOTK01000129.1 Methylobacterium pseudosasicola B NZ VRVH01000094.1:5923-7612 Methylobacterium sp. WL6 NODE 94 length 15034 cov 15.1751 WL6 spades whole genome shotgun sequence 98 M06-LYS072 100 M01-DNA007 100 M06-LYS080 Clade A6 94 50 M01-LYS037 100 M06-DNA012 M01-DNA013 M06-DNA011 NZ VRVF01000010.1:27022-28711 Methylobacterium sp. WL8 NODE 10 length 101617 cov 18.9396 WL8 spades whole genome shotgun sequence NZ LKKO01000001.1 Methylobacterium sp. 78 100 NZ VRUT01000031.1:24152-25841 Methylobacterium sp. WL120 NODE 31 length 34806 cov 12.9269 WL120 spades whole genome shotgun sequence 67 NZ VRVD01001000.1 Methylobacterium sp. 100 NZ VRUX01000130.1:1106-2795 Methylobacterium sp. WL103 NODE 130 length 9324 cov 15.2782 WL103 spades whole genome shotgun sequence NZ VRVG01000100.1 Methylobacterium sp. NZ VRUW01000046.1:19089-20778 Methylobacterium sp. WL12 NODE 46 length 37997 cov 10.4151 WL12 spades whole genome shotgun sequence NZ VRVA01000100.1 Methylobacterium sp. 100 94 100 NZ LSIX01000459.1:15311-16994 Methylobacterium sp. CCH5-D2 CCH5-D2 contig465 whole genome shotgun sequence NZ CP042823.1 Methylobacterium sp. 94 NZ CP029551.1:444986-446675 Methylobacterium sp. 17Sr1-43 chromosome complete genome Clade A7 100 98 NZ VRVI01000100.1 Methylobacterium sp. NZ CABFPH010000016.1:70447-72136 Methylobacterium symbioticum strain SB0023/3 whole genome shotgun sequence M06-DNA020 NZ CABFVH010000031.1:48658-50347 Methylobacterium dankookense strain SW08-7 whole genome shotgun sequence Clade A8 M. komagatae (clade A10) 99 100 M06-DNA021 NZ FOCS01000015.1:127850-129539 Methylobacterium sp. UNC300MFChir4.1 whole genome shotgun sequence 96 NZ LSIX01000001.1 Methylobacterium sp. NZ FOMM01000018.1:127629-129318 Methylobacterium sp. 13MFTsu3.1M2 whole genome shotgun sequence Clade A7 A5 NZ CP029551.1 Methylobacterium sp. NZ FNWP01000015.1:29023-30712 Methylobacterium sp. 275MFSha3.1 whole genome shotgun sequence NZ CABFPH010000001.1 Methylobacterium sp. NZ FOZY01000002.1:29522-31211 Methylobacterium sp. yr668 whole genome shotgun sequence Clade A8 100 100 NZ CABFVH010000001.1 Methylobacterium dankookense 99 NZ SLXZ01000003.1:87031-88720 Methylobacterium sp. BK227 Ga0307702 103 whole genome shotgun sequence 99 NZ VZZK01000001.1 Methylobacterium soli 74 NZ KB911279.1:167663-169352 Methylobacterium sp. 285MFTsu5.1 H288DRAFT scaffold00010.10 whole genome shotgun sequence NZ VZZJ01000001.1 Methylobacterium sp. 100 100 99 NZ FNVI01000012.1:29029-30718 Methylobacterium sp. 190mf whole genome shotgun sequence NZ BJZU01000001.1 Methylobacterium oxalidis Clade A5 NZ BADE01000535.1:9042-10731 Methylobacterium sp. B34 whole genome shotgun sequence NZ CP029550.1 Methylobacterium sp. NZ LMPY01000012.1:301180-302869 Methylobacterium sp. Leaf361 contig 2 whole genome shotgun sequence 96 68 NZ SMNT01000001.1 Methylobacterium sp. NZ LACA01000090.1:28942-30631 Methylobacterium radiotolerans strain RE1.2 contig 90 whole genome shotgun sequence 54 NZ BJZT01000001.1 Methylobacterium haplocladii 100 NZ NKQS01000060.1:77133-78822 Methylobacterium radiotolerans strain MAMP 4754 MP-M-radiotolerans contig 60 whole genome shotgun sequence NZ VRVE01000100.1 Methylobacterium sp. Clade A4 10076 NZ BJXP01000040.1:28705-30394 Methylobacterium radiotolerans strain NBRC 15690 sequence040 whole genome shotgun sequence NZ BJZV01000001.1 Methylobacterium gnaphalii NC 010505.1:968308-969997 Methylobacterium radiotolerans JCM 2831 complete sequence 52 NZ LMQV01000001.1 Methylobacterium sp. 89 97 98 NZ CAJCKR010000014.1:28894-30583 Methylobacterium radiotolerans strain ME94 whole genome shotgun sequence NZ SCOS01000100.1 Methylobacterium sp. NZ SAUO01000001.1:1972417-1974106 Methylobacterium radiotolerans strain ES PA-B5 Ga0193687 11 whole genome shotgun sequence 58 NZ FOSV01000064.1 Methylorubrum salsuginis A6 NZ CP017640.1:1172318-1174007 Methylobacterium sp. C1 chromosome complete genome NZ CP029173.1 Methylobacterium sp. NZ QEKZ01000015.1:28919-30608 Methylobacterium organophilum strain DSM 760 Ga0215672 115 whole genome shotgun sequence 100 NC 010725.1 Methylobacterium populi 97 50 100 NZ NPKS01000008.1:28444-30133 Methylobacterium sp. P1-11 NODE 8 length 205650 cov 35.3187 whole genome shotgun sequence 68 100 NZ LSNC01000001.1 Methylorubrum populi 100 NZ FMWU01000010.1:28770-30459 Methylobacterium sp. UNC378MF whole genome shotgun sequence NZ WEKV01000001.1 Methylorubrum populi 56 NZ FNHS01000013.1:29633-31318 Methylobacterium phyllostachyos strain BL47 whole genome shotgun sequence NZ CP039546.1 Methylorubrum populi NZ VUOK01000062.1:217251-218936 Methylobacterium sp. 2A scaffold7 whole genome shotgun sequence 87 NZ AP014809.1 Methylobacterium populi NZ CP043538.1:4254387-4256072 Methylobacterium mesophilicum SR1.6/6 chromosome complete genome NZ KB316282.1 Methylobacterium sp. NZ JACJIM010000003.1:112963-114652 Methylobacterium fujisawaense strain DSM 5686 Ga0373204 03 whole genome shotgun sequence Clade A9 NZ QJJJ01000001.1 Methylobacterium sp. NZ BACT01000780.1:593-2282 Methylobacterium sp. B1 whole genome shotgun sequence 100 NZ CP021054.1 Methylorubrum zatmanii 56 98 NZ CP016429.1:5748789-5750478 Methylobacterium sp. XJLW chromosome complete genome NZ LT962688.1 Methylobacterium extorquens Clade B 100 53 NZ CP003811.1:1130860-1132549 Methylobacterium oryzae CBMB20 chromosome complete genome NZ BJVP01000001.1 Methylorubrum extorquens NZ FOPK01000010.1:181798-183487 Methylobacterium phyllosphaerae strain CBMB27 whole genome shotgun sequence NC 010172.1 Methylobacterium extorquens 85 50 NZ CP015367.1:462199-463888 Methylobacterium phyllosphaerae strain CBMB27 chromosome complete genome NC 012988.1 Methylobacterium extorquens A7 100 99 NZ JACCCS010000001.1:5219103-5220792 Methylobacterium sp. YL-MPn6-2016 Ga0395893 01 whole genome shotgun sequence NC 011757.1 Methylobacterium extorquens NZ JACCCR010000002.1:99135-100824 Methylobacterium sp. YL-MPn5-2016 Ga0395892 02 whole genome shotgun sequence 99 NZ CP006992.1 Methylobacterium sp. NZ KN050685.1:181353-183040 Methylobacterium sp. UNCCL110 BR90DRAFT scaffold00005.5 whole genome shotgun sequence 100 NZ SRHQ01000001.1 Methylorubrum sp. NZ LMNS01000001.1 Methylobacterium sp. 98 NZ FPBW01000010.1:29940-31627 Methylobacterium sp. UNCCL125 whole genome shotgun sequence 75 NZ AKFK01000046.1:199718-201401 Methylobacterium sp. GXF4 contig49 whole genome shotgun sequence 100 NC 012808.1 Methylobacterium extorquens 73 NZ AGJK01000612.1 Methylorubrum extorquens 100 NZ CP033231.1:501780-503463 Methylobacterium brachiatum strain TX0642 chromosome NZ FOQW01000018.1:91642-93325 Methylobacterium brachiatum strain 111MFTsu3.1M4 whole genome shotgun sequence NZ MNAO01000100.1 Methylorubrum extorquens NZ LHCD01000003.1:82338-84027 Methylobacterium sp. ARG-1 Contig3 whole genome shotgun sequence NZ CP019322.1 Methylorubrum extorquens NZ FOTK01000013.1:127412-129101 Methylobacterium pseudosasicola strain BL36 whole genome shotgun sequence NZ SACP01000100.1 Methylobacterium sp. 97 100 100 NC 010511.1 Methylobacterium sp. NZ LKKO01000019.1:78662-80351 Methylobacterium sp. GXS13 contigs14 whole genome shotgun sequence 99 100 M01-DNA012 NZ KB900609.1 Methylobacterium sp. 100 NZ VRVD01000059.1:12794-14483 Methylobacterium sp. WL18 NODE 59 length 15508 cov 15.3806 WL18 spades whole genome shotgun sequence 80 NC 011894.1 Methylobacterium nodulans NZ VRVG01000004.1:35339-37028 Methylobacterium sp. WL7 NODE 4 length 112908 cov 5.4679 WL7 spades whole genome shotgun sequence NZ SRLB01000001.1 Methylobacterium sp. 99 A8 NZ VRVA01000006.1:143231-144916 Methylobacterium sp. WL64 NODE 6 length 146212 cov 9.95392 WL64 spades whole genome shotgun sequence NZ QOWC01000001.1 Methylobacterium sp. 100 NZ VRVI01000015.1:78718-80403 Methylobacterium sp. WL2 NODE 15 length 81761 cov 11.4706 WL2 spades whole genome shotgun sequence 57 NZ LABY01000001.1 Methylobacterium variabile 100 NZ CP029553.1 Methylobacterium sp. 100 NZ CP042823.1:2368860-2370545 Methylobacterium sp. WL1 chromosome complete genome M01-DNA021 NZ LABX01000001.1 Methylobacterium aquaticum M. komagatae (Clade A10) NZ LABZ01000001.1 Methylobacterium tarhaniae 100 M01-DNA020 58 100 NC 011894.1:1192714-1194403 Methylobacterium nodulans ORS 2060 complete genome NZ CP028843.1 Methylobacterium currus NZ CP029552.1 Methylobacterium sp. NC 010511.1:2412570-2414259 Methylobacterium sp. 4-46 complete sequence 66 Clade C NZ QOWC01000132.1:1882-3571 Methylobacterium crusticola strain MIMD6 contig132 whole genome shotgun sequence 61 NZ FPCB01000083.1 Methylobacterium sp. 94 NZ SACP01000006.1:38196-39863 Methylobacterium sp. TER-1 NODE 6 length 223082 cov 51 whole genome shotgun sequence NZ VDDA01000001.1 Methylobacterium sp. 59 100 69 NZ LABZ01000151.1:594-2277 Methylobacterium tarhaniae strain DSM 25844 contig 151 whole genome shotgun sequence NZ JXOD01000001.1 Methylobacterium platani NZ LABY01000097.1:6675-8358 Methylobacterium variabile strain DSM 16961 contig 97 whole genome shotgun sequence NZ LWHQ01000001.1 Methylobacterium platani 82 100 NZ AP014704.1 Methylobacterium aquaticum NZ PELK01001665.1:5585-7268 Methylobacterium frigidaeris strain IER25-16 C26169 whole genome shotgun sequence 73 99 NZ CP029553.1:1882621-1884304 Methylobacterium terrae strain 17Sr1-28 chromosome complete genome NZ FONO01000081.1 Methylobacterium sp. A9 NZ SRLB01000015.1:171014-172697 Methylobacterium sp. 6HR-1 contig15 whole genome shotgun sequence NZ FOEB01000056.1 Methylobacterium sp. 100 56 NZ LABX01000183.1:10285-11968 Methylobacterium aquaticum strain DSM 16371 contig 183 whole genome shotgun sequence NZ JTHG01000001.1 Methylobacterium platani NZ JAAKGV010000003.1:171135-172818 Methylobacterium sp. DB0501 contig3 whole genome shotgun sequence 78 NZ JTHF01000001.1 Methylobacterium platani NZ LDRM01000001.1 Methylobacterium indicum NZ CP029552.1:4614546-4616229 Methylobacterium sp. 17Sr1-1 chromosome complete genome 100 NZ FPCB01000020.1:146474-148157 Methylobacterium sp. 174MFSha1.1 whole genome shotgun sequence Clade C NZ LDRL01000001.1 Methylobacterium indicum 100 NZ AP014704.1:2150341-2152024 Methylobacterium aquaticum strain MA-22A 95 NZ LDRN01000001.1 Methylobacterium indicum 71 50 NZ FONO01000001.1:80257-81940 Methylobacterium sp. yr596 whole genome shotgun sequence NZ FOEB01000007.1:17130-18813 Methylobacterium sp. ap11 whole genome shotgun sequence Microvirga (2) NZ VDDA01000001.1:384220-385903 Methylobacterium sp. 17Sr1-39 contig1 whole genome shotgun sequence 100 NZ CP028843.1:2534164-2535847 Methylobacterium currus strain PR1016A chromosome 1 Enterovirga 100 NZ LWHQ01000015.1:153538-155221 Methylobacterium platani strain PMB02 contig015 whole genome shotgun sequence NZ JXOD01000105.1:46658-48341 Methylobacterium platani JCM 14648 contig 105 whole genome shotgun sequence 100 NZ JTHF01000106.1:24995-26678 Methylobacterium platani strain SE2.11 contig 106 whole genome shotgun sequence NZ JTHG01000070.1:9958-11641 Methylobacterium platani strain SE3.6 contig 70 whole genome shotgun sequence A10 NZ LDRN01000054.1:10210-11893 Methylobacterium indicum strain NS230 contig 54 whole genome shotgun sequence 99 NZ LDRM01000192.1:10210-11893 Methylobacterium indicum strain NS229 contig 192 whole genome shotgun sequence 100 NZ LDRL01000259.1:10210-11893 Methylobacterium indicum strain NS228 contig 259 whole genome shotgun sequence Microvirga Enterovirga 62 Microvirga 98 C 0.05 0.05 61 Figure S6 - Phylogenetic tree on rpoB partial nucleotide sequences from 187 62 Methylobacterium isolates and 185 reference genomes. 63 Consensus phylogenetic tree from rpoB complete nucleotide sequences (4064 bp) available for 64 153 Methylobacterium genomes and 32 Methylobacteriaceae outgroups (Microvirga, 65 Enterovirga), partial nucleotide sequences from two rpoB HV regions obtained in 20 isolates 66 from the 2017 pilot survey (see Figure S5b; 1,244 bp) and the first rpoB HV region amplified 67 with primers Met02-352-F and Met02-1121-R in 167 isolates from the 2018 timeline survey 68 (700bp; open circles). In the final alignment, sites missing in at least 70% of sequences were 69 removed. Nodes with less than 30% of support were collapsed. The phylogenetic tree was rooted 70 on Microvirga and Enterovirga outgroups. Branch lengths were computed for graphical purpose 71 with the Grafen method. Labels of reference sequences (from complete genomes and pilot survey 72 isolates) as colored according to clade assignation (see Figure S5). 73

7 111MFTsu3.1M4 Methylobacterium brachiatum

TX0642 Methylobacterium brachiatum

WL64 Methylobacterium sp. WL1 Methylobacterium sp. WL2 Methylobacterium sp. WL18 Methylobacterium sp.

GXF4 MethylobacteriumWL7 sp. Methylobacterium sp. ARG

− 1 Methylobacterium sp.

J E J J E J E E

− − − − J − − − −

087 YL 086 048 − 113 128 027 J 022 010 047 019 017 052 033 YL 028 − − E 128 129 − − 066 − − − MPn6 111 148 102 CBMB27 − − − J J − J − E BL36 Methylobacterium pseudosasicola 123 DSM5686 Methylobacterium fujisawaense E 062 J − − MPn5 035 E E − 018 CBMB27 J − 051 J E XJLW Methylobacterium sp. E J − 021 − E − 072 J CBMB20 Methylobacterium− oryzae J − − 121 118 J − 033

O O O J J O 2016 Methylobacterium sp. − O − 034 O 116 − O J − J − O 115 124 O J − O O O − 2016 Methylobacterium sp. J − O E 130 026 O − 2 Methylobacterium phyllosphaerae O E O O J − 115 O − 075 − O E E − O 010 1 Methylobacterium phyllosphaerae 147 O O E − O 028 B1 Methylobacterium sp. J − 012 O J − E O J − 077 − 125 O 011 E − 011 O J − UNCCL110 Methylobacterium sp. O O J − 008 E − 124 O O 100 100 O E UNCCL125 Methylobacterium sp. − 101 O 100 − 285MFTsu5.1 Methylobacterium sp. J O O J 009 − 029 100 O J O 84 O GXS13− Methylobacterium107 sp. E − 120 O 73 O J − 026 070 O 100 31 O J − 002 J − O 100 021 86 O E − UNC300MFChir4.1 Methylobacterium sp. − O 100 O 008 036 O 34 87 O E − 138 O 87 97 100 E − 001 B34 Methylobacterium sp. O 100 51 O J − 107 190mf Methylobacterium sp. O 34 O J 100 O − 104 BK227 Methylobacterium sp. O 100 E − 13MFTsu3.1M2 Methylobacterium sp. O 100 O 024 100 E − O 99 O 141 39 O E − 275MFSha3.1 Methylobacterium sp. 99 O J 074 yr668 Methylobacterium sp. 95 99 O − 133 J − ME94 Methylobacterium radiotolerans 97 100 100 51 O J DSM760 Methylobacterium organophilum 97 O DNA011 Methylobacterium sp. ES PA Leaf361 Methylobacterium sp. 98 O DNA012135 Methylobacterium sp. MAMP4754 Methylobacterium radiotolerans O − 121 100 O J − − 87 E 002 B5 Methylobacterium radiotolerans − JCM2831 Methylobacterium radiotolerans J 100 O LYS080 Methylobacterium sp. 100 O LYS072085 Methylobacterium sp. 46 − 69 O J 066 NBRC15690 Methylobacterium radiotolerans 100 − RE1.2 Methylobacterium radiotolerans J −048 1 Methylobacterium sp. 34 100 E − 17 Methylobacterium sp. 88 O NBRC107715− Methylobacterium oxalidis C1 Methylobacterium sp. O 98 O 17J42 100 34 100 100 17SD2 95 43 47 L1A1 Methylobacterium sp. 32 96 70 Leaf456 Methylobacterium sp. 56 DSM2163 Methylobacterium rhodinum 96 85 UNC378MFP1 2AMethylobacterium Methylobacterium sp. sp. 96 100 CGMCC1.6474 Methylobacterium salsuginis − 11 Methylobacterium sp. 94 89 DB1607 Methylobacterium sp. 100 100 98 MB200 Methylobacterium sp. J− 55 100 DSM5687 Methylobacterium rhodesianum 164 52 CLZ Methylobacterium sp. 98 NI91 Methylobacterium sp. O 10044 DM1 Methylobacterium sp. 58 100 J 100 98 B4 Methylobacterium sp. −064 95 Leaf123 Methylobacterium sp. J 103 −082 100 J− J−127 O 100 31 E O PSBB040 Methylobacterium extorquens −127 O 59 AMS5 Methylobacterium sp. E O 62 −109 100 Q1 Methylobacterium sp. J O −057 98 E−003 E O 98 064 −065 O E− J− O 100 045 O 72 100 99 O RAS18 Methylobacterium sp. E−119 99 CM4 Methylobacterium extorquens E O −114 O 50 NBRC15911 Methylobacterium extorquens J−003 O 82 TK0001 Methylobacterium extorquens J 52 −106 O 97 100 45 PSBB041 Methylobacterium zatmanii J 100 100 PA1 Methylobacterium extorquens −132 O 70 −1 Methylobacterium extorquens E 48 87 DM4 −009 O 83 −2 Methylobacterium extorquens J O 67 37 DM4 −158 100 J−088 O 77 DSM13060 Methylobacterium extorquens 100 85 J−049 O 33 AM1 Methylobacterium sp. 100 100 J−105 O 100 97 J−037 O 100 O −014 J−104 74 99 O J E− O CP3 Methylobacterium extorquens 005 O 68 99 J− 73 J−043 122 O 99 O −1 Methylobacterium sp. E− R2 126 O −XJ1 Methylobacterium populi J−093 63 YC O 100 DSM11490 Methylobacterium thiocyanatum E−046 O 34 96 100 PinkelPinkel 01 Methylobacterium populi E−116 O 39 BL47 Methylobacterium phyllostachyos 0 100 CD11 7 Methylobacterium populi SR1.6/6 Methylobacterium mesophilicum BJ001 Methylobacterium populi 100 P−1M Methylobacterium populi E−006 O 100 100 DSM25903 Enterovirga rhinocerotis J−030 O DB1703 Enterovirga sp. 97 100 81 DNA020 Methylobacterium sp. 33 100 c27j1 Microvirga sp. E−042 O 100 96 100 O 100 17mud1−3 Microvirga sp. J−076 100 HR1 Microvirga sp. J−083 O 100 100 100 100 R24825 Microvirga sp. 100 DSM14364 061 O 37 100 100 DNA021 MethylobacteriumE− sp. O 100 37 DSM21344 Microvirga aerophila J−067 O 100 NBRC106136 Microvirga aerophila J−054 O 100 CDVBN77 Microvirga sp. −039 c23x22 Microvirga sp. E O 100 91 100 J−059 O NCCP 100 −1258 Microvirga pakistanensis J−136 O 100 KCTC23863 Microvirga makkahensis E−063 O 100 BR3299 Microvirga vignae J−073 O 64 KLBC81 Microvirga sp. −005 O 47 CGMCC1.7666 Microvirga guangxiensis J 60 10053 E−106 O R24845 Microvirga sp. −160 O Marseille J 100 100 46 J−029 59 100 CCBAU65841−Q2068 Microvirga Microvirga sp. sp. O WSM3557 Microvirga lotononidis 48 040 O 100 ATCCBAA E− O 134 62 DSM15743 Microvirga flocculans J− O −817 Microvirga flocculans −117 BSC39 Microvirga sp. LYS037 MethylobacteriumJ sp. O 100 100 AT3.9 J−137 O 100 120 99 100 V5/3m Microvirga ossetica E− O 43 99 M8 Microvirga sp. J−152 O 100 020 10034 Lmie10 Microvirga tunisiensis E− O 98 092 O 34 33 SYSUG3D203 Microvirga sp. J− 100 SYSUG3D207 Microvirga sp. −105 O 100 100100 E 016 O 75 100 JC119 − 60 100 MGYG E 156 O 100 − J− 30 JC119 2 Microvirga massiliensis 031 − J− O 62 TER HGUT −089 4 −1 Microvirga massiliensis J O 98 − −1 Methylobacterium− sp. O 66 WSM259846 Methylobacterium Methylobacterium02310 sp. sp.Microvirga massiliensis 025 96 E− O 98 ORS2060 Methylobacterium nodulans −125 O 100 51 6HR J 130 100 100 J− O 40 98 47 DSM16961− Methylobacterium variabile 146 O 17Sr1 1 Methylobacterium sp. J− 100 −015 O DB0501 Methylobacterium sp. J 103 O 100 − 100 100 DSM25844− 28Methylobacterium Methylobacterium tarhaniae sp. DNA007 Methylobacterium sp. E 150 100 DSM16371 Methylobacterium aquaticum J− −065 100 PR1016A Methylobacterium currus J 163 99 − 100 174MFSha1.1 Methylobacterium sp. J 35 17Sr1 O 100 O 68 100 17Sr1 O PMB02 Methylobacterium− platani 46 72 67 100 JCM14648 Methylobacterium1 Methylobacterium platani sp. 100 75 100 100 − 131 O 99 100 SE2.11 Methylobacterium39 Methylobacterium platani sp. − 99 100 J 139 O 79 SE3.6 Methylobacterium platani − 100 J 050 O 100 99 83 NS228 Methylobacterium indicum − O 100100 100 100 NS230 Methylobacterium indicum J O 100 062 100 NS229 Methylobacterium indicum O 100 ap11 Methylobacterium sp. − 022 99 WL93 Methylobacterium sp. J O 65 100 MA − 030 94 99 J − 99 100 96 yr596 Methylobacterium sp. WL116 Methylobacterium sp. E 112 100 84 MIMD6 Methylobacterium− sp. − 041 100 22A Methylobacterium aquaticum E 47 100 NBRC107716 Methylobacterium gnaphalii WL119 Methylobacterium sp. − 142 100 DSM24105 Methylobacterium brachythecii WL30 Methylobacterium sp. E − 94 66 J 037 100 100 NBRC107714 Methylobacterium haplocladii − 100 100 100 O WL9 Methylobacterium sp. E 100 59 60 67 CCH5 96 100 49 O 17Sr1 31 54 85 SW08 WL6 Methylobacterium sp. 37 74 SB0023/3 Methylobacterium sp. 34 E 100 YIM132548 Methylobacterium sp.− O 100 100 − YIM48816 Methylobacterium soli− D2 Methylobacterium sp. O O 123 J − 43 Methylobacterium sp. O Leaf93− Methylobacterium sp. 77 Methylobacterium sp. 7 Methylobacterium dankookense Leaf91 Methylobacterium047 sp. O O Leaf86 Methylobacterium sp. O LYS093 Methylobacterium sp. O LYS069 Methylobacterium sp. E 10 Methylobacterium sp. O O 090 O Leaf106 Methylobacterium− sp. 162 J − 88A Methylobacterium sp. − WL19 Methylobacterium− sp. 045 J Leaf85 Methylobacterium sp. J J J 112 WL8 Methylobacterium sp. Leaf465 Methylobacterium sp. Leaf94 Methylobacterium sp. − Leaf89 Methylobacterium sp. Leaf88 Methylobacterium sp. − Leaf111 Methylobacterium sp. GV104 Methylobacterium sp. GV094 Methylobacterium sp. Leaf104 Methylobacterium sp. LYS051 Methylobacterium sp. 078 084 068

153 WL120 Methylobacterium sp. −

J −

108 056 WL103 Methylobacterium sp. 132 WL12 Methylobacterium sp. J

− − −

J J

E DNA013 Methylobacterium sp. Leaf469 Methylobacterium sp. Leaf102Leaf87 Methylobacterium Methylobacterium sp. sp.

Leaf112 Methylobacterium sp. Leaf100 Methylobacterium sp. Leaf99 MethylobacteriumWL69 Methylobacterium sp. sp.

105 Methylobacterium gossipiicolaDNA018 Methylobacterium sp. − Leaf117 Methylobacterium sp. BTF04 Methylobacterium sp. Leaf113 Methylobacterium sp. Gh Leaf108 Methylobacterium sp. Leaf466 Methylobacterium sp. Leaf399 Methylobacterium sp. V23 Methylobacterium sp. Leaf125 Methylobacterium sp. LYS027 Methylobacterium sp.

LYS083 Methylobacterium sp. DNA006 Methylobacterium sp.

DNA014 Methylobacterium sp.

DNA010 Methylobacterium sp. DNA001 Methylobacterium sp.

DNA024 Methylobacterium sp. 74 Figure S7 - Iterative normalization of the rpoB phylogeny for graphical purpose. The rpoB 75 phylogenetic tree (Figure S6) was normalized so it was scaled proportionally to nucleotide 76 pairwise similarity (PS). a) Level in the tree (log(L)) in function of modified pairwise similarity 77 (PSc= -log(1.005-PS)) before iteration (initial tree). b) Pearson’s correlation coefficient (r2) 78 between log(L) and PSc during 10,000 scaling iterations. c) log(L) in function of PSc in the 79 scaled tree (after 10,000 iterations). d) PS in function of level (L) in the scaled tree. 80

8 before iterations iterations −0.6 2.2

2.0 −0.7

1.8

−0.8 1.6 r2

modified PS 1.4 r2 = −0.6071 −0.9 1.2

1.0 −1.0

2.5 3.0 3.5 4.0 4.5 5.0 5.5 0 500 1000 1500 2000 2500 3000

Level (log) Iteration after iterations real values

1.00 2.2

0.98 2.0

1.8 0.96

1.6 PS 0.94 modified PS 1.4 r2 = −0.9948 0.92 1.2

0.90 1.0

4.0 4.5 5.0 5.5 50 100 150 200 250 300

Level (log) Level 81 Figure S8 - Analysis of diversity in positive controls (METH communities) before ASVs 82 filtering and rarefaction. a) pairwise comparison in ASV absolute abundance between METH 83 replicates (across 4 sequencing runs). b) Determination of a conservative threshold to distinguish 84 true ASVs (from known rpoB nucleotide sequences of isolates from which genomic DNA were 85 mixed together to generate the METH community) from false ASVs (contaminant, sequencing 86 and PCR errors, chimeric ASVs). 87

9

88 Figure S9 - ASV rarefaction on diversity assessed by rpoB barcoding in 184 phyllosphere 89 samples, 4 positive controls (METH community) and 4 negative controls. a) Rarefaction 90 curves (Black = phyllosphere samples; red= METH communities; blue= negative controls). b) 91 Cumulated ASVs relative abundance per sample before rarefaction, from the most abundant to 92 the rarest ASV. c) Cumulated ASVs relative abundance per sample after rarefaction, from the 93 most abundant to the rarest ASV (negative control excluded). 94

10

95 Figure S10 - Diversity within METH communities after rarefaction. a) ML phylogenetic tree 96 (100 permutations, complete deletion, Tamura-Nei model) of partial rpoB nucleotide sequence 97 (410 bp) retrieved from 10 reference genomes (squares; including Sphingomonas sp.), amplified 98 by SANGER sequencing in 18 Methylobacterium isolates used to built the METH community 99 (full circles), the 18 corresponding true Methylobacterium ASVs and one likely true 100 Sphingomonas ASV (open circle) and 25 false ASVs resulting from contamination from 101 phyllosphere samples (c). b) ASV relative abundance in METH communities. 102

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103 Figure S11 - Unrooted ML phylogenetic tree based on rpoB partial nucleotide sequences 104 from 1,344 ASVs (410 bp). Only nodes supported by at least 50% of bootstraps (200 105 permutations) are shown. ASVs are labeled according to their taxonomic assignation based on 106 rpoB nucleotide sequence database (legend on bottom left). 107

12

108 Figure S12 - Unrooted ML phylogenetic tree based on rpoB partial nucleotide sequences 109 from 283 Methylobacteriaceae ASV (points) and 232 references isolates and genomes 110 (unlabeled tips). Only nodes supported by at least 50% of bootstraps (200 permutations) are 111 shown. Full circle indicate 200 Methylobacterium ASVs assigned to clades (colored) when 112 clustering with identified reference sequences with at least 50% of support. Unassigned ASVs are 113 indicated in grey. 114

13

115 Figure S13 - Comparison of Methylobacterium diversity assessment from rpoB barcoding 116 and isolation. Number of Methylobacterium isolates in function of ASV relative abundance 117 assuming a) exact sequence match between ASVs and isolates; b) 98.5% of sequence identity 118 (maximum of 6 nucleotide mismatches). c) Comparison of Methylobacterium clade relative 119 abundances in phyllosphere estimated from available reference genomes (black), 16S sequencing 120 (community barcoding: pale red; sanger on isolates from the pilot survey: red) and rpoB 121 sequencing (community barcoding: pale blue; sanger on timeline survey isolates: blue). 122

14

123 Figure S14 - Experimental design of Methylobacterium monitoring for growth performance 124 under four temperature treatments. a) 79 isolates (two showed in this example: pink and cyan) 125 and two negative controls (not showed) were tested for ability to grow under different 126 temperature treatments. b) Pre-conditioning step (P): For each isolate and negative controls and 127 each temperature treatment (20 and 30 °C), 10µL of cellular culture from stock were spread on 128 solid Methanol-MMS media. c) After 20 days of incubation at 20 °C (P20) or 30 °C (P30), petri

129 dishes were swabbed and collected cell concentrations adjusted to OD630=0.2. d) Monitoring step 130 (M): Each pre-conditioned culture P20 (n=81) and P30 (n=81), was spotted on new Methanol- 131 MMS media (five replicates per culture, per P treatment). Each petri dish was duplicated, one 132 copy for incubation at 20 °C (M20 treatment) and one for incubation at 30 °C (M30). Pictures of 133 petri dishes were took 7, 13 and 24 after inoculation. e) Three examples of spot organization on 134 petri dishes (24 per M treatment, 17 isolates + one negative control per dish). Open circle 135 represent negative controls. 136

15 a b c d e

P20 M20 P20M20 P30M20 20°C x5 x80

Strain 1 x24 20 d. 7 d. 6 d. 11 d. P20M30 P30M30

Strain 2

30°C x5 P30 M30 x24 137 Figure S15 - Example of image analysis of Methylobacterium monitoring for growth 138 performance under four temperature treatments. a) Original picture. b) The original picture 139 was converted in grey scale in ImajeJ. Areas outside of the agar, as well as every visible particle 140 other than bacteria spot within the agar area, were manually cropped (black). The picture was 141 duplicated. A copy was used for background correction (BACK; bacteria spots cropped). Another 142 copy was used to measure raw bacteria spot intensities (BW). c) Reconstruction of background 143 intensities. d) Correction of raw intensities by subtracting background values. e) Definition of 144 growth area: detail of a spot (top) and average pixel intensities in function of the radius of 145 concentric circles drawn from the center of the colony. Growth area is defined as the circle with 146 maximal T value in t-test comparison between intensities outside and within the area (here in 147 red). f) Comparison of intensity distributions outside (red; local background) and within (blue; 148 spot) growing area. Dotted lines indicate average intensity values. g) Border effect: spot 149 intensities after correction for local background (I) are shown for 48 petri dishes and 3 time 150 points in function of their average position of each petri. Because of less competition for 151 nutrients, I values (proportional to point size) are in average higher close to the border of the petri 152 dish (spot positioned according to the original picture). h) Expected I values (scale on top) in 153 function of X/Y position on the petri dish predicted from a polynomial regression 2 2 2 2 154 (I∼X Y +X Y+XY +X+Y). i) Corrected I values (residuals from the polynomial regression). 155

16

156 Figure S16 - Prediction of log normal growth curve, growth rate and yield for 79 isolates 157 incubated under four temperature treatments. a) Log normal best prediction for four different 158 observed cases (legend on bottom). Curves were predicted in the range 0-36 days form values

159 observed at T7, T13 and T24, assuming null intensity at T0. Models assuming that intensity

160 remained null until Ti were tested in the range 0-7 days. b) determination of yield (Y, maximum 161 intensity) and growth rate (r = 1/log+lag) from predicted growth curve. c) comparison of 162 predicted yield and maximum observed intensity. d) comparison of predicted log+lag values with

163 time (T7, T13 or T24) at which maximum intensity was observed. e) All predicted growth curves 164 showed separately for each temperature treatment. Replicates for which maximum intensity was 165 not reach at day 36 according to the model were discarded (log+lag≥36). 166

17

167 REFERENCES 168 1. Tamura K, Nei M. 1993. Estimation of the number of nucleotide substitutions in the 169 control region of mitochondrial DNA in humans and chimpanzees. Mol Biol Evol 10:512–526. 170 2. Kumar S, Stecher G, Tamura K. 2016. MEGA7: Molecular Evolutionary Genetics 171 Analysis Version 7.0 for Bigger Datasets. Mol Biol Evol 33:1870–1874. 172 3. Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. 2016. 173 DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods 13:581– 174 583. 175

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