SUPPLEMENTAL MATERIAL Physiology of Genotypes of Coral-Associated Endosymbionts

Home , Bacteroidetes, Cytophagales, Hyphomonadaceae, Roseivivax

SUPPLEMENTAL MATERIAL

Physiology of Genotypes of Coral-Associated Endosymbionts (Breviolum antillogorgium) in

Response to Bacterial Communities

Chelsea Aline Brisson

Master of Science in Biology

SUPPLEMENTARY TABLES

Table T1) Pairwise post hoc contrasts of H’ alpha diversity metrics using emmeans in both experiments. Significant differences within and between experiments are bold.

Experiment 4x4 Experiment Temp-Env

Bacterial

Community 16-0583 16-0584 16-0630 16-0732 16-0583 16-0630 16-0723

16-0584 0.9994 ------

16-0630 0.5113 0.78 - - - - - 4x4

Experiment 16-0732 0.2253 0.1119 0.0121 - - - -

16-0583 1 0.9911 0.377 0.3214 - - -

16-0630 0.9912 1 0.8178 0.0515 0.9498 - -

Env -

16-0723 1 1 0.5853 0.0983 0.9974 0.9994 -

Temp Experiment Environmental 0.9757 0.8271 0.1595 0.6479 0.9967 0.6408 0.8617

Table T2) Taxa in Experiment 1 that significantly differ in relative abundance between genotypes – results from ANCOM analysis (Qiime2). Taxa shown are only those that are significantly different enough to reject the null hypothesis that the relative abundance is the same among all genotypes (W statistic). Phylogenetic Taxa W Level Class Bacteria – Spirochaetes – Leptospirae 16 Order Bacteria – Bacteroidetes – Bacteroidia – unknown 72 Family Bacteria – Bacteroidetes – Bacteroidia – Cytophagales – 102 Amoebophilaceae Genus Bacteria – Bacteroidetes – Bacteroidia – Cytophagales – 137 Amoebophilaceae – Uncultured

Table T3) Taxa in initial/inoculum communities from Experiment 2 that significantly differ in relative abundance between genotypes – results from ANCOM analysis (Qiime2). Taxa shown are only those that are significantly different enough to reject the null hypothesis that the relative abundance is the same among all genotypes (W statistic).

Phylogenetic Taxa W Level Class Bacteroidetes – Rhodothermia 20 Class Planctomycetes – Phycisphaerae 17 Order Proteobacteria – Alphaproteobacteria – Sphingomonadales 80 Order Proteobacteria – Alphaproteobacteria – Thalassobaculales 77 Family Proteobacteria – Alphaproteobacteria – Sphingomonadales – 126 Sphingomonadaceae Family Proteobacteria – Alphaproteobacteria – Thalassobaculales – 122 Thalassobaculaceae Family Proteobacteria – Alphaproteobacteria – Rhizobiales – Stappiaceae 116 Family Proteobacteria – Alphaproteobacteria – Rhodospirillales – 115 Terasakiellaceae Genus Proteobacteria – Alphaproteobacteria – Sphingomonadales – 191 Sphingomonadaceae – Altererythrobacter Genus Proteobacteria – Alphaproteobacteria – Thalassobaculales – 186 Thalassobaculaceae – Thalassobaculum

Table T4) Pairwise PERMANOVA tests of all final bacterial communities in Experiment Temp- Env. Communities were tested at the genus level using the (a) Bray-Curtis metric and (b) binary- Jaccard metric. Communities were grouped by which bacterial community they were inoculated with and tested against all other communities. Bold denotes a significant p-value. Values below the diagonal are p-values; above the diagonal have a Bonferroni correction. Values on the diagonal are the initial bacterial community composition tested against any final samples inoculated with that community.

(a) Initial Community

16-0583 16-0630 16-0723 Environmental

y 16-0583 0.252 0.028 1 0.028 16-0630 0.001 0.42 0.028 0.028

Final Final 16-0723 0.126 0.001 0.28 0.028

Communit Environmental 0.001 0.001 0.001 1

(b) Initial Community 16-0583 16-0630 16-0723 Environmental

16-0583 0.196 0.028 1 0.028 ty 16-0630 0.001 0.056 0.028 0.026

Final Final 16-0723 0.094 0.001 0.168 0.028

Communi Environmental 0.001 0.001 0.001 0.812 Table T5) Summary table of ANOVA results of the effects of microbiome, host genotype, and temperature on taxa that changed the most from initial to final community. These are separated by (a) phyla, (b) families within the class Alphaproteobacteria, (c) families within the class Gammaproteobacteria, and (d) families within the phyla Bacteroidetes.

(a) Phyla: Bacteroidetes Proteobacteria Planctomycetes Factor F value P value F value P value F value P value Algal Genotype 1.521 0.23596 0.247 0.7828 0.659 0.5253 Bacterial Community 5.590 0.00391 1.380 0.2694 0.286 0.8354 Temperature 4.829 0.03643 0.472 0.4976 4.474 0.0434 Genotype : Bacteria 2.498 0.04614 0.964 0.4671 1.066 0.4060 Genotype : Temperature 0.653 0.52843 0.045 0.9558 1.301 0.2882 Bacteria : Temperature 8.008 0.00178 2.544 0.0866 0.524 0.5976 3-way Interaction 0.213 0.80985 2.017 0.1519 3.014 0.0652

(b) Family: Rhizobiales Rhodobacterales Sphingomonadales Caulobacterales Factor F value P value F value P value F value P value F value P value Algal Genotype 0.199 0.8209 2.579 0.09376 1.652 0.20981 1.823 0.1802 Bacterial Community 2.074 0.1262 10.607 7.88*10-5 33.713 1.96*10-9 14.77 5.9*10-6 Temperature 0.652 0.4261 0.543 0.46716 9.000 0.00562 0.2407 0.2407 Genotype : Bacteria 0.104 0.9953 2.264 0.06604 1.818 0.13164 3.129 0.0179 Genotype : Temperature 0.213 0.8091 0.298 0.74471 0.507 0.60796 0.014 0.9866 Bacteria : Temperature 2.683 0.0859 0.934 0.40481 11.878 0.00018 0.706 0.5023 3-way Interaction 0.368 0.6952 9.479 0.00072 0.517 0.60209 0.004 0.9959 (c) Family: Alteromonadales Cellvibrionales Oceanospirillales Salinisphaerales Factor F value P value F value P value F value P value F value P value Algal Genotype 2.804 0.0776 6.777 0.00398 1.248 0.3027 1.634 0.21328 Bacterial Community 74.836 1.74*10-13 15.238 4.53*10-6 1.510 0.2335 5.506 0.00422 Temperature 5.018 0.0332 5.940 0.02141 0.060 0.8077 0.026 0.87347 Genotype : Bacteria 3.260 0.0147 7.449 7.80*10-5 0.833 0.5545 2.708 0.03351 Genotype : Temperature 5.846 0.0076 7.744 0.00210 0.153 0.8593 0.591 0.56058 Bacteria : Temperature 9.378 0.0008 4.636 0.01823 4.999 0.0139 0.149 0.86187 3-way Interaction 2.588 0.0931 9.215 8.41*10-4 1.628 0.2144 0.772 0.47145 (d) Family: Balneolales Cytophagales Flavobacteriales OPB56 Factor F value P value F value P value F value P value F value P value Algal Genotype 4.899 0.015 0.402 0.67272 0.857 0.4353 0.444 0.646 Bacterial Community 0.461 0.711 0.983 0.41495 4.155 0.0148 1.10532 <2.0*10-16 Temperature 0.566 0.458 0.219 0.64324 3.157 0.0865 0.024 0.878 Genotype : Bacteria 1.101 0.386 2.232 0.06941 0.713 0.6425 0.640 0.697 Genotype : Temperature 1.353 0.275 1.713 0.00229 0.030 0.9702 0 1.0 Bacteria : Temperature 0.179 0.837 7.614 0.19869 2.276 0.1214 0 1.0 3-way Interaction 0.149 0.862 0.746 0.48325 0.015 0.9847 0 1.0

Table T6) P-values from Spearman’s Rank Correlation tests of the most abundant bacteria at each phylogenetic level vs each physiological trait at ambient (26C) temperature. Bold signifies a significant p-value. Taxa are listed in order of average relative abundance (greatest to least) for each phylogenetic level. GROWTH RATE Class Order Family Genus Alphaproteobacteria 0.693 Rhizobiales 0.3998 Rhodobacteraceae 0.5022 Pseudohongiella 0.0927 Gammaproteobacteria 0.2665 Rhodobacterales 0.5022 Hyphomonadaceae 0.0544 Labrenzia 0.4268 Bacteroidia 0.8438 Oceanospirillales 0.8915 Pseudohongiellaceae 0.0927 Algimonas 0.0220 Rhodothermia 0.6158 Caulobacterales 0.0544 Stappiaceae 0.4268 Roseivivax 0.9821 Phycisphaerae 0.1889 Cytophagales 0.0721 Halomonadaceae 0.4814 Marinobacter 0.7043 PHOTOSYNTHETIC EFFICIENCY Class Order Family Genus Alphaproteobacteria 0.1076 Rhizobiales 0.159 Rhodobacteraceae 0.1136 Pseudohongiella 0.982 Gammaproteobacteria 0.0595 Rhodobacterales 0.1136 Hyphomonadaceae 0.6734 Labrenzia 0.3652 Bacteroidia 0.6623 Oceanospirillales 0.3554 Pseudohongiellaceae 0.982 Algimonas 0.6067 Rhodothermia 0.8867 Caulobacterales 0.6734 Stappiaceae 0.3652 Roseivivax 0.2903 Phycisphaerae 0.8035 Cytophagales 0.6623 Halomonadaceae 0.7918 Marinobacter 0.124 CHLOROPHYLL A Class Order Family Genus Alphaproteobacteria 0.1081 Rhizobiales 0.917 Rhodobacteraceae 0.0522 Pseudohongiella 0.4133 Gammaproteobacteria 0.1458 Rhodobacterales 0.0522 Hyphomonadaceae 0.7043 Labrenzia 0.3215 Bacteroidia 0.7156 Oceanospirillales 0.1702 Pseudohongiellaceae 0.4133 Algimonas 0.493 Rhodothermia 0.5322 Caulobacterales 0.7043 Stappiaceae 0.3215 Roseivivax 0.2171 Phycisphaerae 0.5408 Cytophagales 0.253 Halomonadaceae 0.3396 Marinobacter 0.0667 PHOTOSYNTHESIS RATE Class Order Family Genus Alphaproteobacteria 0.1119 Rhizobiales 0.4265 Rhodobacteraceae 0.2334 Pseudohongiella 0.5781 Gammaproteobacteria 0.0439 Rhodobacterales 0.2334 Hyphomonadaceae 0.8676 Labrenzia 0.6097 Bacteroidia 0.253 Oceanospirillales 0.0069 Pseudohongiellaceae 0.5781 Algimonas 0.2717 Rhodothermia 0.5733 Caulobacterales 0.8676 Stappiaceae 0.6097 Roseivivax 0.2913 Phycisphaerae 0.5008 Cytophagales 0.8676 Halomonadaceae 0.3007 Marinobacter 0.0459 RESPIRATION RATE Class Order Family Genus Alphaproteobacteria 0.208 Rhizobiales 0.4591 Rhodobacteraceae 0.0164 Pseudohongiella 0.0599 Gammaproteobacteria 0.8343 Rhodobacterales 0.0164 Hyphomonadaceae 0.2732 Labrenzia 0.5699 Bacteroidia 0.5886 Oceanospirillales 0.7936 Pseudohongiellaceae 0.0599 Algimonas 0.8889 Rhodothermia 0.1195 Caulobacterales 0.2732 Stappiaceae 0.5699 Roseivivax 0.0880 Phycisphaerae 0.1156 Cytophagales 0.3254 Halomonadaceae 0.0455 Marinobacter 0.4268

Table T7) P-values from Spearman’s Rank Correlation tests of the most abundant bacteria at each phylogenetic level vs each physiological trait at elevated (30C) temperature. Bold signifies a significant p-value. Taxa are listed in order of average relative abundance (greatest to least) for each phylogenetic level. GROWTH RATE Class Order Family Genus Alphaproteobacteria 0.6584 Rhizobiales 0.5264 Rhodobacteraceae 0.9663 Pseudohongiella 0.0186 Gammaproteobacteria 0.3446 Rhodobacterales 0.9663 Hyphomonadaceae 0.5451 Labrenzia 0.3876 Bacteroidia 0.1256 Oceanospirillales 0.0327 Pseudohongiellaceae 0.0186 Algimonas 0.7837 Rhodothermia 0.9016 Caulobacterales 0.5562 Stappiaceae 0.3876 Roseivivax 0.3314 Phycisphaerae 0.159 Cytophagales 0.8229 Halomonadaceae 0.0015 Marinobacter 0.1856 PHOTOSYNTHETIC EFFICIENCY Class Order Family Genus Alphaproteobacteria 0.8674 Rhizobiales 0.328 Rhodobacteraceae 0.9541 Pseudohongiella 0.3574 Gammaproteobacteria 0.7126 Rhodobacterales 0.9541 Hyphomonadaceae 0.5118 Labrenzia 0.1646 Bacteroidia 0.8132 Oceanospirillales 0.3168 Pseudohongiellaceae 0.3574 Algimonas 0.7958 Rhodothermia 0.4471 Caulobacterales 0.6334 Stappiaceae 0.1646 Roseivivax 0.3686 Phycisphaerae 0.9271 Cytophagales 0.3157 Halomonadaceae 0.8422 Marinobacter 0.1654 CHLOROPHYLL A Class Order Family Genus Alphaproteobacteria 0.7173 Rhizobiales 0.122 Rhodobacteraceae 0.3103 Pseudohongiella 0.1824 Gammaproteobacteria 0.18 Rhodobacterales 0.3103 Hyphomonadaceae 0.1046 Labrenzia 0.1662 Bacteroidia 0.4959 Oceanospirillales 0.2762 Pseudohongiellaceae 0.1824 Algimonas 0.1665 Rhodothermia 0.0149 Caulobacterales 0.135 Stappiaceae 0.1662 Roseivivax 0.0339 Phycisphaerae 0.09828 Cytophagales 0.8564 Halomonadaceae 0.121 Marinobacter 0.5899 PHOTOSYNTHESIS RATE Class Order Family Genus Alphaproteobacteria 0.3652 Rhizobiales 0.9237 Rhodobacteraceae 0.5072 Pseudohongiella 0.8427 Gammaproteobacteria 0.5528 Rhodobacterales 0.5072 Hyphomonadaceae 0.0952 Labrenzia 0.8344 Bacteroidia 0.3381 Oceanospirillales 0.4796 Pseudohongiellaceae 0.8427 Algimonas 0.0823 Rhodothermia 0.3497 Caulobacterales 0.1157 Stappiaceae 0.8344 Roseivivax 0.8026 Phycisphaerae 0.6365 Cytophagales 0.1711 Halomonadaceae 0.7092 Marinobacter 0.3602 RESPIRATION RATE Class Order Family Genus Alphaproteobacteria 0.0223 Rhizobiales 0.0275 Rhodobacteraceae 0.6324 Pseudohongiella 0.0965 Gammaproteobacteria 0.0144 Rhodobacterales 0.6324 Hyphomonadaceae 0.0342 Labrenzia 0.0109 Bacteroidia 0.0048 Oceanospirillales 0.1854 Pseudohongiellaceae 0.0965 Algimonas 0.2224 Rhodothermia 0.517 Caulobacterales 0.0252 Stappiaceae 0.0109 Roseivivax 0.1203 Phycisphaerae 0.1012 Cytophagales 0.0007 Halomonadaceae 0.6098 Marinobacter 0.3003

SUPPLEMENTARY FIGURES

(a)

(b)

Figure F1) PCoA plots of the initial bacterial communities from Experiment 4x4 at the (a) order level and (b) family level. Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Dissimilarity matrices were made using the binary Jaccard metric.

(a) (b)

(e)

Figure F2) PCoA plots of the initial bacterial communities from Experiment 4x4 at the (a) class level, (b) order level, (c) family level, (d) genus level, and (e) species level. Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Dissimilarity matrices were made using the Bray-Curtis metric.

(a)

(b)

(c)

Figure F3) PCoA plots of the initial bacterial communities from Experiment 4x4 using the (a) binary Jaccard, (b) Bray-Curtis, and (c) unweighted unifrac distances between ASVs (amplicon sequence variants). Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Plots were generated by QIIME2.

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Figure F6) PCoA plots of the initial bacterial communities from Experiment Temp-Env at the (a) class level, (b) order level, (c) family level, and (d) genus level. Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Dissimilarity matrices were made using the binary Jaccard metric. (a) (b)

(e)

Figure F7) PCoA plots of the initial bacterial communities from Experiment 4x4 at the (a) class level, (b) order level, (c) family level, (d) genus level, and (e) species level. Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Dissimilarity matrices were made using the Bray-Curtis metric.

(a)

(b)

Figure F8) PCoA plots of the initial bacterial communities from Experiment Temp-Env using the (a) binary Jaccard and (b) Bray-Curtis distances between ASVs (amplicon sequence variants). Colors denote the endosymbiont genotype from which the bacterial communities were extracted. Plots were generated by QIIME2.

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Figure F11) RDA of final communities in experiment 2 the (a) class, (b) order, and (c) genus levels, plotted against physiological traits. Longer arrows indicate stronger associations.

Figure F12) Change in relative abundance of top most changed phyla in experiment 2. Difference calculated from relative abundances of initial-final.

(a)

(b)

Figure F13) Change in relative abundance of top most changed Alphaproteobacteria orders in experiment 2 in an (a) heatmap and (b) separates change by temperature, genotype, and bacterial community. Difference calculated from relative abundances of initial-final.

(a)

(b)

Figure F14) Change in relative abundance of top most changed Gammaproteobacteria orders in experiment 2 in an (a) heatmap and (b) separates change by temperature, genotype, and bacterial community. Difference calculated from relative abundances of initial-final. (a)

(b)

Figure F15) Change in relative abundance of top most changed Bacteroidetes orders in experiment 2 in an (a) heatmap and (b) separates change by temperature, genotype, and bacterial community. Difference calculated from relative abundances of initial-final.

Figure S18) Relative abundance of all phyla in final communities in experiment 2. Empty bars represent missing samples.

Figure S19) Average relative abundance of classes in final bacterial communities in experiment 2. Other category is the sum of all classes less than 1% relative abundance. Empty bars represent missing samples.

Figure S20) Average relative abundance of families in final bacterial communities in experiment 2. Other category is the sum of all families less than 1% relative abundance. Empty bars represent missing samples.