New Phytologist Supporting Information Article title: Species-specific Root Microbiota Dynamics in Response to Plant-Available Phosphorus Authors: Natacha Bodenhausen, Vincent Somerville, Alessandro Desirò, Jean-Claude Walser, Lorenzo Borghi, Marcel G.A. van der Heijden and Klaus Schlaeppi Article acceptance date: Click here to enter a date. The following Supporting Information is available for this article: Figure S1 | Analysis steps Figure S2 | Comparison of ITS PCR approaches for plant root samples Figure S3 | Rarefaction curves for bacterial anD fungal OTU richness Figure S4 | Effects of plant species anD P-levels on microbial richness, diversity anD evenness Figure S5 | Beta-diversity analysis including the soil samples Figure S6 | IDentification of enDobacteria by phylogenetic placement Table S1 | Effects of plant species anD P treatment on alpha Diversity (ANOVA) Table S2 | Effects of plant species anD P treatment on community composition (PERMANOVA) Table S3 | Effects P treatment on species-specific community compositions (PERMANOVA) Table S4 | Statistics from iDentifying phosphate sensitive microbes Table S5 | Network characteristics MethoDs S1 | Microbiota profiling anD analysis Notes S1 | Comparison of PCR approaches Notes S2 | Bioinformatic scripts Notes S3 | Data analysis in R Notes S4 | Mapping enDobacteria Notes S5 | Comparison of ITS profiling approaches Alpha diversity all samples Rarefaction analysis Figure S3 (vegan) Raw counts plant samples Rarefication (500x) to Figure S4 ANOVA 15’000 seq/sample Table S1 Filter: OTUs with min. 4 counts in min. 4 samples Normalization by all samples PCoA, Filtered counts total sum scaling (TSS) PERMANOVA plant Figure S5, Table S2 plant samples samples Differential abundance testing PCoA, PERMANOVA Normalization by Table S4 (edgeR) trimmed mean of M Taxonomic Figure 4, Table S3 Identification of P values (TMM) composition sensitive OTUs Figure 2 Beta diversity (phyloseq, vegan) Spearman rank Display network graph Data display with ternary correlation and Figure 6, Table S5 (igraph) plots and boxplots module detection Co-occurrence analysis Figure 5 Figure S1 | Analysis steps The schematic flow diagram illustrates the steps of the analysis in R. Individual types of normalization steps, analyses or statistical tests are indicated with the blue boxes. Larger grey boxes segment the analysis and indicate the major R-packages that were used in alpha- and beta diversity analyses, differential abundance testing and network analysis. Analysis outputs (Figures and Tables) are indicated in red at their respective analysis steps. Figure S2 | Comparison of ITS PCR approaches for plant root samples To profile the root-associated fungal communities of Petunia, we first evaluated three ITS PCR approaches to test whether they avoid co-amplification of plant ITS sequences and whether they permit a reliable quantification of Glomeromycotina fungi. Four root DNA extracts from Petunia growing under low P conditions (=heavily colonized by AMF) were amplified with ITS1F and ITS2 by McGuire et al. (2013), fITS7 and ITS4 by Ihrmark et al. (2012) and the ITS1F with the reverse complement of fITS7. (a) The illustration depicts the positions of the PCR primers amplifying the internal transcribed spacer (ITS) regions between the small- and large ribosomal sub-units (SSU/LSU) of the ribosomal operon. Separate community profiles were produced and inspected for the proportions of plant and AMF sequences as well as for fungal diversity. Taxonomic composition of the 4 replicate extracts is reported at the level of Domain (b) and within the fungi at the level of detected Phyla (c). (D) The diversity captured by the PCR approaches was determined by rarefying the fungi data and recording OTU richness. Bars represent means (n = 100; ± s.e.m.) and letters indicate groups differing significantly at P < 0.05 (Tukey’s HSD). Bacteria 2000 Soil Petunia Arabidopsis 1500 1000 number of fOTUs number 500 0 0 10000 20000 30000 40000 50000 60000 70000 Sample Size Fungi 700 Soil Petunia 600 Arabidopsis 500 400 300 200 number of bOTUs number 100 0 0 10000 20000 30000 40000 50000 60000 70000 Sample Size Figure S3 | Rarefaction curves for bacterial anD fungal OTU richness We conducted a sampling intensity analysis for bacteria (a) and fungi (b) with all samples (Arabidopsis and Petunia with reddish and blueish colors, respectively, and the increasing P-levels (low, medium to high) are marked with increasing hue. Random sub-samplings were conducted for sequencing depths in steps of 100 sequences with 1000 iterations per sequencing depth. The average number of detected OTUs is reported for each sampling depth. The black vertical line indicates the selected rarefaction depth (15,000 sequences) used statistical analysis of alpha diversity (Fig. S4). Figure 4 Alpha diversity S: *** T: *** SxT: S: *** T: * SxT: *** Arabidopsis Petunia Arabidopsis Petunia 1000 A AB B A AB B 300 B A A A A A ● ● ● ●● ● ● ● ●● ●● ● ●●● ● ●● ● ● ●● ● ● ●●● 800 200 ●●● ● ● ● ● ● ●● ● ● ●● ● ● ● ● ● ●● ● ● ● ● ●● ● ● fOTU richness fOTU ●● bOTU richness bOTU ● ●● 100 600 ● ● ●● ●●●● ●●●● ● ● ●● ●● ●●● ●● ●● ● ●● low medium high low medium high low medium high low medium high S: *** T: ** SxT: S: *** T: SxT: Arabidopsis Petunia Arabidopsis Petunia A A A A B B A A A A A A ● ●● ● 120 ●● ● ● 20 ● ● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● ●●● ●● ●● 80 ●● ● ●● ● ● ● 10 ●● ●● fOTU diversity fOTU bOTU diversity bOTU ●● ● ● 40 ●● ●●● ●● ● ●●● ● ● ●● ●●●●●●●●● ● ● ●●●●●●● ●●●●●●● 0 0 low medium high low medium high low medium high low medium high S: *** T: ** SxT: S: *** T: . SxT: * Arabidopsis Petunia Arabidopsis Petunia A A A A AB B 0.125 A B B A A A ● ● 0.15 ● ●● ● 0.100 ● ● ● ●● ●● ● ● ●● ● ●● ● ● ●● ●●● ●● ● 0.075 ●● 0.10 ●● ●●● ●● ● ● ● ●● ●● 0.050 ● ● ●●● ● ● evenness fOTU ● bOTU evenness bOTU ● ● ●● 0.05 ● ● ● ●● ● ● 0.025 ●● ● ●●●●●● ●●●● ● ● ●● ●●●●●●●● low medium high low medium high low medium high low medium high Figure S4 | Effects of plant species anD P-levels on microbial richness, diversity anD evenness Alpha diversity was assessed based on OTU richness, Shannon diversity and Sheldon evenness on data with a common sequencing depth of 15’000 sequences per sample. ANOVA was used to test for species- (S), treatment- (T) or their interaction (SxT) effects and their level of significance is indicated above plots (P < 0.001 ***; P < 0.01 **; P < 0.05 *; Table S1 contains the details of this ANOVA). Different letters indicate significant pairwise differences between different levels of P availability (P < 0.05, Tukey HSD). Figure S4 Beta diversity (with soil) Bacteria Fungi ●● ●● ● ●● ● ●● ●●● ● ● ●●● ●●● ● ● 0.1 ● ● ●●●● ●● 0.25 ● ● ● ● 0.0 ●● ● ●●● ●●●● ● ● ● ● ● ● ● ● ●● ●●● ● ● ● ●●● ● ●●● ●● 0.00 ●● ● ● ●●●●● ● ● ● PCo 2 (9.2%) ● ● ● ●● ● PCo 2 (23.9%) −0.1 ●●● ●● ● ●● ● ● ● ●● ●●●●●● ● ● ●●●● ● ● ● ● ●●● −0.25 ●●● −0.2 ● ● −0.2 0.0 0.2 0.4 −0.4 −0.2 0.0 0.2 PCo 1 (45.8%) PCo 1 (52.1%) Figure S5 | Beta-diversity analysis including the soil samples Principal coordinate analysis using Bray-Curtis dissimilarities were performed to investigate effects of plant species and P-levels on community composition. (a) Bacterial and (b) fungal communities associated with Arabidopsis roots (reddish colors), with Petunia roots (blueish colors) and found in unplanted soil (brownish colors), all sampled from varying levels of P availability (low, medium to high, marked with increasing hue). bOTU 1020 bOTU 1046 BRE from Mortierella elongata FMR13-2, AB558494 BRE from Mortierellomycotina Burkholderia bOTU 1682 Uncultured bacterium, KC663974 bOTU 33 endobacteria Uncultured bacterium, EF019895 Environmental BRE bOTU 390 Mycoavidus cysteinexigens B1-EB, LC005489 * M. cysteinexigens from Mortierella elongata AG77, KP772716 bOTU 1995 Mycoavidus cysteinexigens from Mortierellomycotina M. cysteinexigens from Mortierella humilis PMI1414, KP772723 M. cysteinexigens from Mortierella elongata AG67, KP772725 CaGg from Scutellospora pellucida BR208A, JF816842 CaGg from Scutellospora pellucida BR208A, JF816849 - bOTU 134 related BRE CaGg from Gigaspora margarita BEG34, X89727 CaGg from Scutellospora castanea BEG1, AJ251636 CaGg from Gigaspora margarita CM23, KF378649 Candidatus Glomeribacter gigasporarum CaGg from Gigaspora margarita CM47, KF378652 from Glomeromycotina CaGg from Gigaspora margarita MAFF520054, KF378648 * CaGg from Gigaspora margarita BR444, JF816839 CaGg from Scutellospora pellucida CL750A, JF816850 Betaproteobacteria CaGg from Scutellospora pellucida FL704, JF816857 bOTU 2298 Burkholderia endofungorum, AM420302 Burkholderia endofungorum, HQ005412 Burkholderia rhizoxinica, AJ938142 Burkholderia rhizoxinica, HQ005411 Burkholderia pseudomallei, AY305818 Burkholderia mallei, NR041725 Burkholderia oklahomensis, NR118070 * Burkholderia cepacia, AY741362 Burkholderia graminis, NR029213 74 Burkholderia phytofirmans, NR042931 bOTU 84 Burkholderia mimosarum, HE864347 Pandoraea apista, NR028749 Pandoraea terrae, NR152002 Pseudoduganella sp., KU647209 bOTU 11 Duganella sp., KU647207 Pseudoduganella danionis, NR152711 Herbaspirillum huttiense, EU046556 Herbaspirillum aquaticum, FJ267649 Uncultured bacterium, AB487893 bOTU 2334 Uncultured bacterium, JX394270 Herbaspirillum sp., AB769222 Herbaspirillum sp., AB542410 * bOTU 1822 99 Ralstonia pickettii, NR043152 bOTU 529 Ralstonia solanacearum, CP012939 Cupriavidus basilensis, CP010536 Cupriavidus metallidurans, CP000352 Thiobacillus thioparus, GU967681 bOTU 915 Thiobacillus sajanensis, DQ390445 Spirillum winogradskyi, AY845251 Spirillum