Supplementary materials:

Diversity and abundance of Bacteria and nirS-encoding denitrifiers associated with the Juan de Fuca Ridge hydrothermal system

Annals of Microbiology

Annie Bourbonnaisa, b, 1, S. Kim Junipera, David A. Butterfieldc,d, Rika E. Andersone, and Moritz F. Lehmannf a School of Earth and Ocean Sciences, University of Victoria, Victoria, BC V8P 5C2, Canada bnow at: School for Marine Science and Technology (SMAST), University of Massachusetts Dartmouth, New Bedford, MA 02744-1221, USA cJoint Institute for the Study of the Atmosphere and Ocean, University of Washington, Seattle, WA 98105-5672, USA dPacific Marine Environmental Laboratory, National Oceanic and Atmospheric Administration, Seattle, WA 98115, USA eSchool of Oceanography and Astrobiology, University of Washington, Seattle, WA 98195-7940, USA fDepartment of Environmental Sciences, University of Basel, 4056 Basel, Switzerland 1Author to whom correspondence should be addressed: [email protected] Table S1. Similarity coefficients used to compare 16S rRNA gene V1-V3 tag sequences and nirS gene bacterial community richness, membership and structure in hydrothermal vent fluids sampled at two different vent fields (Endeavour Segment and Axial Volcano) on the JFR. The percentage of shared sequences is indicated in brackets. Sample names as in Table 1. Richnessa Membershipb Structurec

Sobs, chao1 and Jclass Sor Jest Sor Jabund Sor the- the- ACE class est abund ayc tan

16S rRNA gene AV07BC/F 94 (37%) 0.94 0.88 0.93 0.88 0.86 0.76 0.95 0.87 AV07BC/S 10 (14%) 0.99 0.98 0.99 0.98 0.98 0.97 0.98 0.98 AV07BC/ 74 (37%) 0.96 0.92 0.96 0.92 0.90 0.81 0.92 0.90 TS AV07BC/ 91 (32%) 0.95 0.90 0.95 0.90 0.87 0.77 0.98 0.89 ES09H2 AV07BC/ 18 (11%) 0.99 0.98 0.99 0.98 0.99 0.98 1.00 0.99 ES09P AV07F/S 14 (13%) 0.98 0.96 0.98 0.96 0.96 0.92 0.96 0.96 AV07F/TS 143 (70%) 0.88 0.78 0.88 0.78 0.60 0.43 0.74 0.63 AV07F/ 70 (32%) 0.95 0.90 0.94 0.89 0.87 0.77 0.99 0.89 ES09H2 AV07F/ 37 (25%) 0.97 0.93 0.95 0.91 0.95 0.90 0.99 0.96 ES09P AV07S/TS 19 (20%) 0.98 0.96 0.98 0.97 0.95 0.90 0.97 0.95 AV07S/ 10 (6.3%) 0.99 0.98 0.99 0.98 0.98 0.96 1.00 0.99 ES09H2 AV07S/ 11 (8.9%) 0.99 0.97 0.99 0.97 0.97 0.94 1.00 0.97 ES09P ES09H2/P 111 (22%) 0.91 0.84 0.90 0.82 0.72 0.56 0.88 0.78 AV07TS/ 94 (40%) 0.94 0.89 0.94 0.88 0.88 0.79 0.98 0.90 ES09H2 AV07TS/ 48 (54%) 0.97 0.93 0.96 0.93 0.96 0.92 0.99 0.97 ES09P nirS gene AV07S- ES09H1 1 (59%) 0.95 0.90 0.97 0.94 0.58 0.41 0.25 0.58 a. Number of shared OTUs observed (Sobs) and shared richness estimates from the Chao1 (SChao1) and ACE (SACE) estimators for an OTU definition (97% identical sequences). b. Jaccard (J) and Sorenson (Sor) similarity coefficients based on the observed richness (class) or the Chao1 estimated richness (est). The maximum value of 1 indicates that samples are entirely dissimilar. c. Abundance-based Jaccard (Jabund) and Sorenson (Sorabund) similarity coefficients and Yue and Clayton theta (theayc) and Smith theta (thetan) similarity coefficients. The scale ranges from 1 (entirely dissimilar) to 0 (identical). Table S2. P-values from P-test (Martin et al., 2002) and UniFrac distance metric (Lozupone and Knight, 2005) implemented in Fast-UniFrac to compare 16S rRNA gene (V1-V3 regions) bacterial communities in hydrothermal vent fluids of the JFR. The p-values have been corrected using the Bonferroni correction. Significance is indicated by an asterisk next to the p-value. Sample names as in Table 1. AV07BC AV07F AV07S AV07TS ES09H2 P-test AV07BC AV07F <=1.0e-03* AV07S <=1.0e-03* <=1.0e-03* AV07TS <=1.0e-03* <=1.0e-03* <=1.0e-03* ES09H2 <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* ES09P <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* UniFrac/ un- weighted AV07BC AV07F <=1.0e-03* AV07S <=1.0e-03* <=1.0e-03* AV07TS <=1.0e-03* 1 <=1.0e-03* ES09H2 <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* ES09P <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* <=1.0e-03* UniFrac/ weighted AV07BC AV07F 0.33 AV07S <=1.0e-03* <=1.0e-03* AV07TS <=1.0e-03* 1 <=1.0e-03* ES09H2 <=1.0e-03* 1 <=1.0e-03* 1 ES09P <=1.0e-03* 0.18 <=1.0e-03* 0.045* 0.03* Figure S1. UPGMA dendrogram comparing the similarity of 16S rRNA gene bacterial communities at six diffuse vent fluids of the JFR based on the Yue and Clayton theta similarity coefficient at the 3% difference level as calculated in MOTHUR. The length of the scale bar represents a distance of 0.1. Sample names as in Table 1. 0.5 0.5 0.5 a) b) c) 0.4 0.4 0.4 ES09P AV07F ES09P 0.3 0.3 0.3

0.2 AV07TS 0.2 AV07TS 0.2 AV07TS

0.1 0.1 0.1 ES09H2 AV07F ES09H2 AV07F 0 0 AV07BC 0

-0.1 -0.1 AV07S -0.1

-0.2 -0.2 ES09P -0.2

-0.3 -0.3 -0.3 AV07BC AV07S ES09H2 AV07SAV07BC -0.4 -0.4 -0.4 0 0.1 0.2 0.3 0.4 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 -0.4 -0.3 -0.2 -0.1 P1 (25.84%) P1 (25.84%) P3 (18.53%) 0.5 0.4 0.5 d) 0.4 e) f) AV07BC 0.3 0.4 AV07BC

0.3 0.3 0.2 AV07F 0.2 0.2 0.1 ES09H2 AV07TS 0.1 0.1 AV07F 0 AV07F 0 ES09H2 AV07S 0 ES09H2 AV07TS AV07TS AV07S -0.1 -0.1 -0.1 AV07S -0.2 ES09P -0.2 AV07BC -0.2

-0.3 -0.3 -0.3 ES09P -0.4 -0.4 -0.4 ES09P 0.2 0.4 0.6 0.8 1.0 1.0 -0.4 -0.2 0 -0.4 -0.2 0 0.2 0.4 0.6 0.8 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 P1 (63.74%) P1 (63.74%) P3 (8.33%) Figure S2. Comparison of 16S rRNA gene bacterial communities obtained from pyrosequencing of the variable V1-V3 regions in hydrothermal vent fluids at Axial Volcano (blue) and Endeavour Segment (red) on the JFR. The scatter-plots were generated using the un-weighted (a, b, c) and weighted (d, e, f) UniFrac matrix of pairwise distances between communities and Principal Coordinate Analysis (PCoA). The three main components (explaining 67.2% and 91.2% of the variance for the un-weighted and weighted abundance analysis, respectively) are presented. Sample names were assigned as in Table 1. The percent of variation explained for each component is indicated in brackets. Figure S3. Relative abundance (in %) of nirS genes grouping in different clusters of the phylogenetic tree (Fig. 4) in hydrothermal vent fluids of a) Endeavour Segment (Hulk1) and b) Axial Volcano (Shepherd) (see Table 2).