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1 Supplementary Figures 1 2 Fine-Scale Adaptations To 1 SUPPLEMENTARY FIGURES 2 3 FINE-SCALE ADAPTATIONS TO ENVIRONMENTAL VARIATION AND GROWTH 4 STRATEGIES DRIVE PHYLLOSPHERE METHYLOBACTERIUM 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 Microvirga (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 bacteria 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
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