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Evidence for Strain-Specific Exometabolomic Responses of The Emiliania huxleyi exometabolome – Supplementary Material Evidence for strain-specific exometabolomic responses of the coccolithophore Emiliania huxleyi to grazing by the dinoflagellate Oxyrrhis marina Supplementary Material Kelsey L. Poulson-Ellestad,1† Elizabeth L. Harvey,2‡ Matthew D. Johnson,2 and Tracy J. Mincer1* 1Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA USA 2Department of Biology, Woods Hole Oceanographic Institution, Woods Hole, MA USA *Correspondence: Dr. Tracy J. Mincer Woods Hole Oceanographic Institution, 226 Woods Hole Road, MS#51, Woods Hole, MA, 02536, USA [email protected] †Present Address: Department of Biology, Chemistry and Physical Sciences, Roosevelt University, Chicago, IL, USA ‡Present Address: Department of Marine Sciences, Skidaway Institute of Oceanography, University of Georgia, Savannah, GA, USA 1 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Figure 1. Mean growth rate of O. marina fed four different strains of E. huxleyi. Growth rates (µ) were averaged across all concentrations of E. huxleyi tested. Error bars represent ±1 S.D. Letters (a-b) indicate statistical equivalency from ANOVA with Tukey post- test (n = 10-13). 2 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Figure 2: PCA Scores plot of exometabolomes from four different E. huxleyi strains, either grown alone (circles) or exposed to O. marina grazers grazed (triangles). Letters “A-E” indicate different concentrations of E. huxleyi, with concentration “A” the lowest and concentration “E” the highest. Dashed line indicates 95% confidence interval. 3 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Figure 3. A) Chromatographic comparison of example E. huxleyi exometabolome (black trace) and authentic standard of 4-methyl-5-thiazoleethanol (red trace). Shown is the total ion count. 4-methyl-5-thiazoleethanol peak with retention time at 5 minutes is indicated with an asterisk. B) High resolution mass spectra of 4-methyl-5-thiazoleethanol (red, top) and same feature in exometabolome of CCMP379 (bottom, black). 4 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Figure 4. PCA scores plot comparing DHB607 and DHB624 exometabolomes when normalized to volume culture extracted (left panels) cell concentration (right panels). For strain DHB624 (top panels), there is a significant separation between grazed and control extracts along PC1 for both methods. For strain DHB607 (bottom panels) there is a significant separation between grazed and control extracts along PC3 and PC4 when normalized to volume extracted (left panel). When normalized to the number of E. huxleyi cells (right panel), there is a small but significant separation between grazed and control extracts along PC2. 5 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Table 1: 2-way ANOVA results from PC1 scores E. huxleyi CCMP374. Factors were the presence of O. marina grazers ("Grazing") and E. huxleyi cell concentration ("Cell Conc."). Source of SS DF MS F (DFn, DFd) P value Variation Grazing 176.6 1 176.6 F (1, 12) = 447.7 P < 0.0001 Cell Conc. 0.7354 3 0.2451 F (3, 12) = 0.6215 P = 0.6145 Grazing x Cell 6.547 3 2.182 F (3, 12) = 5.533 P = 0.0128 Conc. Residual 4.734 12 0.3945 Total 188.616 6 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Table 2. Chemical features detected in exometabolomes of E. huxleyi strain CCMP374 Detected in Retention Loading Predator Prey Predator Time on PC1 Only Only Only Feature # m/z (min) Fold Change FDR<0.05 (37.6%) (t = 0 h) (t = 0 h) (t = 48 h) 505 74.0615 2.6 2.57 0.035 0.085 yes yes yes 518b 118.1226 16.9 -1.25 0.029 -0.166 yes yes yes 123b 122.0579 16.0 -1.91 0.033 -0.155 yes yes yes 232a 144.0479 5.4 2.55 0.0077* 0.300 yes yes yes 318a 181.1085 2.4 2.99 0.0038* 0.306 yes yes yes 134 215.1256 14.8 1.70 0.037 0.080 yes yes yes 73 222.1827 20.8 -0.55 0.048 -0.059 yes yes yes 243b 244.2631 19.8 -1.82 0.025* -0.160 yes yes yes 532a 249.1349 5.4 3.29 0.015* 0.234 yes yes yes 341 251.1500 6.2 Grazed Only 0.019* 0.167 no no no 344a 265.1299 5.1 6.36 0.013* 0.240 yes yes yes 326a 265.1300 4.1 3.33 0.0019* 0.314 yes yes yes 519 279.1604 21.2 -0.38 0.05 -0.041 yes yes yes 351a 281.1612 7.2 4.11 0.0112* 0.279 yes yes yes 76a 297.1019 4.1 3.03 0.0058* 0.298 yes yes yes 312 305.1573 12.0 5.37 0.044 0.086 yes yes yes 522a 329.1104 12.3 Grazed Only 0.021* 0.223 no no no 530b 337.1984 19.1 -1.06 0.031 -0.160 yes yes yes 304 357.2745 18.0 9.55 0.046 0.083 yes yes no 511b 399.2340 19.1 -2.54 0.027* -0.162 no no no 517a 400.1467 10.4 Grazed Only 0.023* 0.208 no no no 51 413.2668 21.0 -0.48 0.038 -0.092 yes yes yes 309 495.1493 13.1 9.10 0.042 0.086 yes yes no 317 539.1753 13.5 7.13 0.04 0.086 yes yes yes a indicates features with increased concenrations in response to grazing (PC1 loadings > 0.2) b indicates features with decreased concentrations in response to grazing (PC1 loadings < -0.15) * Indicates significant discovery after FDR correction 7 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Table 3: 2-way ANOVA results from analysis of PC2 scores E. huxleyi CCMP379. Factors were the presence of O. marina grazers ("Grazing") and E. huxleyi cell concentration ("Cell Conc."). Source of SS DF MS F (DFn, DFd) P value variation Grazing 64.09 1 64.09 F (1, 16) = 20.51 P = 0.0003 Cell Conc. 84.8 4 21.2 F (4, 16) = 6.785 P = 0.0022 Grazing x Cell 44.4 4 11.1 F (4, 16) = 3.552 P = 0.0295 Conc. Residual 49.99 Total 243.28 8 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Table 4. Chemical features detected in exometabolomes of E. huxleyi strain CCMP379 Detected in Retention Loading on Predator Prey Predator Predator Time Selectivity PC 2 Only Only + Prey Only Feature # m/z (min) Fold Change FDR<0.05 Ratio (32.22%) (t = 0 h) (t = 0 h) (t = 0 h ) (t = 48 h) 359 100.1118 4.0 -4.16 0.011 0.16 -0.142 no no no no 201b 120.0807 5.6 -1.71 0.029 0.10 -0.156 yes yes yes yes 210b 132.2061 3.3 -2.54 0.018 0.13 -0.164 no no yes no 221b 132.2380 3.3 -2.61 0.016 0.13 -0.159 no no no no 324b 133.1050 3.3 -1.86 0.021 0.11 -0.158 yes yes yes yes 71a 139.0753 14.6 0.06 0.050 0.00 0.194 yes yes yes yes 21a 144.0480 5.1 0.14 0.045 0.01 0.201 yes yes yes yes 368a 146.0444 5.1 0.21 0.044 0.01 0.221 yes yes yes yes 382b 155.1542 10.8 -3.14 0.013 0.14 -0.154 no no yes no 64a 157.0859 14.5 0.12 0.048 0.01 0.195 yes yes yes yes 220b 158.1175 5.8 -2.65 0.015 0.13 -0.169 no no no no 118b 166.0864 5.6 -1.71 0.026 0.11 -0.155 yes yes yes yes 207b 171.1482 17.3 -3.57 0.010 0.23 -0.172 no no no no 189a 181.1089 2.4 0.67 0.023 0.12 0.172 yes yes yes yes 383b 208.0966 15.1 -1.64 0.034 0.10 -0.179 yes yes yes yes 264 227.1064 16.0 Grazed Only 0.008 0.74 0.075 no no no no 262b 231.1702 3.3 -1.01 0.039 0.05 -0.160 yes yes yes yes 386b 247.1288 3.2 -1.82 0.031 0.10 -0.150 no no no no 83a 251.1508 5.9 0.64 0.035 0.09 0.249 yes yes yes yes 215b 261.1444 3.7 -2.37 0.019 0.12 -0.157 no no yes no 358a 265.1294 4.7 0.77 0.032 0.09 0.239 yes yes yes yes 192a 265.1295 3.9 0.44 0.037 0.05 0.204 yes yes yes yes 18a 267.1455 2.8 0.54 0.024 0.10 0.225 yes yes yes yes 351 268.1485 2.8 0.32 0.042 0.02 0.137 yes yes yes yes 193a 282.1154 7.1 1.37 0.006 0.37 0.220 yes yes yes yes 115a 297.1015 3.9 2.01 0.002* 1.93 0.250 yes yes yes yes 360 309.2046 20.0 -5.76 0.005* 0.78 -0.069 yes yes yes yes 340 357.2734 17.9 0.59 0.047 0.01 0.057 no yes no no 339 359.2896 18.5 6.02 0.040 0.10 0.033 yes no yes no a indicates features with increased concentrations in response to grazing (PC2 loadings > 0.15) b indicates feature with decreased concentrations in response to grazing (PC2 loadings < - 0.15) * significant discovery after FDR correction 9 Emiliania huxleyi exometabolome – Supplementary Material Supplementary Table 5.
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