Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis)
Abstract
Faecal metabarcoding is widely used for mammalian diet analysis. However, most extraction protocols are designed to target high molecular weight genomic DNA and not the short DNA sequences associated with digested prey items. To examine the prey composition in southern right whale (Eubalaena australis) faecal samples we trialled a phosphate buffer DNA extraction method along with two commercial extraction kits (QIAamp Fast DNA Stool Mini and QIAGEN DNEasy PowerSoil) with the following variations: 1) incubation time in a phosphate buffer (1 and 24 hours); 2) processing both pellet and supernatant from the phosphate buffer incubations; and 3) two different concentrations of DNA binding buffer. We found that the choice of extraction protocol influenced the richness, diversity and composition of eukaryotes (18S rDNA) and crustaceans (Crust16S mtDNA) in the faecal samples. The PowerSoil protocol performed well for both markers, delivering the highest target richness for 18S rDNA and highest diversity for Crust16S mtDNA, with the pellet of the phosphate buffer also performing comparably. Taxonomic composition in the phosphate buffer supernatant was influenced by the incubation period and concentration of binding buffer and differed from its corresponding pellet. To maximise taxonomic coverage, we recommend combining the extracts from both the supernatant and pellet. In faecal studies, our findings reinforce the importance of defining the community attributes (richness versus diversity versus composition) of key interest prior to performing DNA extraction, as the inference of these variables is likely to be altered by the choice of extraction protocol.
Full text
Supplementary material associated with Optimizing DNA extraction protocols for the diet analysis of a baleen whale (Eubalaena australis) Aashi Parikh1, Richard O’Rorke2, Emma L. Carroll2, Els Vermeulen3, Robert Harcourt1, Anthony Chariton1 1School of Natural Sciences, Macquarie University, Wallumattagal Campus, Macquarie Park NSW 2109 Australia 2School of Biological Sciences, Waipapa Taumata Rau – University of Auckland, Auckland 1142 New Zealand 3Mammal Research Institute Whale Unit, Faculty of Natural and Agricultural Sciences, University of Pretoria, Hatfield 0028 South Africa
Table S1. List of SRW samples used in this study and associated metadata. Sample Date collected Location Storage SA-1 24/10/2006 South Africa – St Helena Bay Ethanol SA-2 Unknown South Africa – St Helena Bay Ethanol SA-3 10/12/1992 South Africa – St Helena Bay Ethanol NZ-1 15/8/2017 New Zealand – Auckland Islands Frozen NZ-2 8/8/2017 New Zealand – Auckland Islands Frozen Table S2. PCR conditions for 18S rDNA and Crrust16S mtDNA. Amplicon PCR Mixture (20 uL) PCR Conditions PCR Stage Temperature Time PCR cycles 18S 2x Amplitaq Gold 360 Mastermix Initial denaturation 95°C 8 min Primer-F (0.1mM) Denaturation 95°C 20 s x 43 Primer-R (0.1mM) Annealing 58°C 20 s PNA clamp (0.5mM)* Elongation 72°C 30 s DNA (2uL) Final elongation 72°C 8 min Crust16S 2x Amplitaq Gold 360 Mastermix Initial denaturation 95°C 10 min Primer-F (0.2mM) Denaturation 95°C 30 s x 45 Primer-R (0.2mM) Annealing 51°C 30 s DNA (2uL) Elongation 72°C 45 s Final elongation 72°C 10 min *Whale blocking PNA clamp: CGACCGTCTTCTCAGC-Lys Table S3. Classifiaction of target and non-target organisms captured by 18S rDNA and Crust16S mtDNA. Target Non-Target 18S rDNA Kingdom Metazoa (free-floating marine zooplankton within this kingdom) Amoebozoa, Cryptophyceae, Discoba, Metamonada, Opisthokonta, Rhodophyta, Sar and Viridiplantae
Class Metazoa: Polychaeta, Hexanauplia, Malacostraca, Ostracoda, Thecostraca, Sagittoidea, Ascidiacea, Cnidaria, Hydrozoa, Scyphozoa, Ctenophora, Lophotrochozoa, Bivalvia, Gastropoda, Protostomia, Eurotatoria Metazoa: Palaeacanthocephala, Clitellata, Arachnida, Collembola, Insecta, Cestoda and Trematoda Crust16S mtDNA Class Metazoa: Malacostraca, Thecostraca Metazoa: Arachnida, Collembola, Eurotatoria, Gymnolaemata, Hydrozoa, Insecta, Mammalia Table S4. Summary of target zOTU richness captured by each extraction protocol for the 18S rDNA dataset. Extraction protocol Mean zOTU richness SD Ph1P.1B 3.8 3.25 Ph1P.2B 3.67 2.28 Ph1S.1B 3.04 2.67 Ph1S.2B 3.27 3.11 Ph24P.1B 4.16 3.75 Ph24P.2B 4.16 3.7 Ph24S.1B 2.58 3.53 Ph24S.2B 3.24 2.81 Soil.1B 4.6 3.8 Soil.2B 4.73 4.37 Stool.1B 4.69 2.79 Stool.2B 3.51 2.65 Table S5. Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the 18S rDNA dataset. Protocols Estimate Std Error z value Pr(>|z|) Ph1P.2B-Ph1P.1B -0.13333 0.4486 -0.297 1 Ph1S.1B-Ph1P.1B -0.75556 0.4486 -1.684 0.8758 Ph1S.2B-Ph1P.1B -0.53333 0.4486 -1.189 0.9899 Ph24P.1B-Ph1P.1B 0.35556 0.4486 0.793 0.9997 Ph24P.2B-Ph1P.1B 0.35556 0.4486 0.793 0.9997 Ph24S.1B-Ph1P.1B -1.22222 0.4486 -2.725 0.2146 Ph24S.2B-Ph1P.1B -0.55556 0.4486 -1.238 0.9859 Soil.1B-Ph1P.1B 0.8 0.4486 1.783 0.8278
Soil.2B-Ph1P.1B 0.93333 0.4486 2.081 0.6369 Stool.1B-Ph1P.1B 0.88889 0.4486 1.981 0.7065 Stool.2B-Ph1P.1B -0.28889 0.4486 -0.644 1 Ph1S.1B-Ph1P.2B -0.62222 0.4486 -1.387 0.9662 Ph1S.2B-Ph1P.2B -0.4 0.4486 -0.892 0.9992 Ph24P.1B-Ph1P.2B 0.48889 0.4486 1.09 0.9952 Ph24P.2B-Ph1P.2B 0.48889 0.4486 1.09 0.9952 Ph24S.1B-Ph1P.2B -1.08889 0.4486 -2.427 0.3883 Ph24S.2B-Ph1P.2B -0.42222 0.4486 -0.941 0.9987 Soil.1B-Ph1P.2B 0.93333 0.4486 2.081 0.6368 Soil.2B-Ph1P.2B 1.06667 0.4486 2.378 0.4217 Stool.1B-Ph1P.2B 1.02222 0.4486 2.279 0.4923 Stool.2B-Ph1P.2B -0.15556 0.4486 -0.347 1 Ph1S.2B-Ph1S.1B 0.22222 0.4486 0.495 1 Ph24P.1B-Ph1S.1B 1.11111 0.4486 2.477 0.355 Ph24P.2B-Ph1S.1B 1.11111 0.4486 2.477 0.3546 Ph24S.1B-Ph1S.1B -0.46667 0.4486 -1.04 0.9968 Ph24S.2B-Ph1S.1B 0.2 0.4486 0.446 1 Soil.1B-Ph1S.1B 1.55556 0.4486 3.468 0.0258 * Soil.2B-Ph1S.1B 1.68889 0.4486 3.765 <0.01 ** Stool.1B-Ph1S.1B 1.64444 0.4486 3.666 0.0134 * Stool.2B-Ph1S.1B 0.46667 0.4486 1.04 0.9968 Ph24P.1B-Ph1S.2B 0.88889 0.4486 1.981 0.7056 Ph24P.2B-Ph1S.2B 0.88889 0.4486 1.981 0.707 Ph24S.1B-Ph1S.2B -0.68889 0.4486 -1.536 0.9309 Ph24S.2B-Ph1S.2B -0.02222 0.4486 -0.05 1 Soil.1B-Ph1S.2B 1.33333 0.4486 2.972 0.1163 Soil.2B-Ph1S.2B 1.46667 0.4486 3.269 0.0499 * Stool.1B-Ph1S.2B 1.42222 0.4486 3.17 0.0671 . Stool.2B-Ph1S.2B 0.24444 0.4486 0.545 1 Ph24P.2BPh24P.1B 0 0.4486 0 1 Ph24S.1BPh24P.1B -1.57778 0.4486 -3.517 0.0221 * Ph24S.2BPh24P.1B -0.91111 0.4486 -2.031 0.6727 Soil.1B-Ph24P.1B 0.44444 0.4486 0.991 0.9979 Soil.2B-Ph24P.1B 0.57778 0.4486 1.288 0.9808 Stool.1B-Ph24P.1B 0.53333 0.4486 1.189 0.9899 Stool.2B-Ph24P.1B -0.64444 0.4486 -1.437 0.9563 Ph24S.1BPh24P.2B -1.57778 0.4486 -3.517 0.0223 * Ph24S.2BPh24P.2B -0.91111 0.4486 -2.031 0.6724
Soil.1B-Ph24P.2B 0.44444 0.4486 0.991 0.9979 Soil.2B-Ph24P.2B 0.57778 0.4486 1.288 0.9807 Stool.1B-Ph24P.2B 0.53333 0.4486 1.189 0.9899 Stool.2B-Ph24P.2B -0.64444 0.4486 -1.437 0.9563 Ph24S.2BPh24S.1B 0.66667 0.4486 1.486 0.9446 Soil.1B-Ph24S.1B 2.02222 0.4486 4.508 <0.01 *** Soil.2B-Ph24S.1B 2.15556 0.4486 4.805 <0.01 *** Stool.1B-Ph24S.1B 2.11111 0.4486 4.706 <0.01 *** Stool.2B-Ph24S.1B 0.93333 0.4486 2.081 0.6366 Soil.1B-Ph24S.2B 1.35556 0.4486 3.022 0.1025 Soil.2B-Ph24S.2B 1.48889 0.4486 3.319 0.0426 * Stool.1B-Ph24S.2B 1.44444 0.4486 3.22 0.0578 . Stool.2B-Ph24S.2B 0.26667 0.4486 0.594 1 Soil.2B-Soil.1B 0.13333 0.4486 0.297 1 Stool.1B-Soil.1B 0.08889 0.4486 0.198 1 Stool.2B-Soil.1B -1.08889 0.4486 -2.427 0.3871 Stool.1B-Soil.2B -0.04444 0.4486 -0.099 1 Stool.2B-Soil.2B -1.22222 0.4486 -2.725 0.2141 Stool.2B-Stool.1B -1.17778 0.4486 -2.625 0.265 Table S6. Summary of target zOTU richness captured by each extraction protocol for the Crust16S mtDNA dataset. Extraction protocol Mean zOTU richness SD Ph1P.1B 8.31 5 Ph1P.2B 6.31 5.22 Ph1S.1B 5.33 3.05 Ph1S.2B 7.6 6.06 Ph24P.1B 7.58 6.02 Ph24P.2B 7.6 5 Ph24S.1B 3.82 3.68 Ph24S.2B 5.73 2.67 Soil.1B 7.18 4.3 Soil.2B 6.62 7.15 Stool.1B 4.07 4.23 Stool.2B 4.98 3.35 Table S7. Post hoc pairwise comparison of differences in target zOTU richness derived from different protocols from the Crust16S mtDNA dataset. Protocols Estimate Std Error z value Pr(>|z|) Ph1P.2B-Ph1P.1B -2 0.99471 -2.011 0.6869 Ph1S.1B-Ph1P.1B -2.97778 0.99471 -2.994 0.1109
Ph1S.2B-Ph1P.1B -0.71111 0.99471 -0.715 0.9999 Ph24P.1BPh1P.1B -0.73333 0.99471 -0.737 0.9999 Ph24P.2BPh1P.1B -0.71111 0.99471 -0.715 0.9999 Ph24S.1BPh1P.1B -4.48889 0.99471 -4.513 <0.01 *** Ph24S.2BPh1P.1B -2.57778 0.99471 -2.591 0.2845 Soil.1B-Ph1P.1B -1.13333 0.99471 -1.139 0.9929 Soil.2B-Ph1P.1B -1.68889 0.99471 -1.698 0.8698 Stool.1B-Ph1P.1B -4.24444 0.99471 -4.267 <0.01 ** Stool.2B-Ph1P.1B -3.33333 0.99471 -3.351 0.038 * Ph1S.1B-Ph1P.2B -0.97778 0.99471 -0.983 0.9981 Ph1S.2B-Ph1P.2B 1.28889 0.99471 1.296 0.9799 Ph24P.1BPh1P.2B 1.26667 0.99471 1.273 0.9825 Ph24P.2BPh1P.2B 1.28889 0.99471 1.296 0.9798 Ph24S.1BPh1P.2B -2.48889 0.99471 -2.502 0.3382 Ph24S.2BPh1P.2B -0.57778 0.99471 -0.581 1 Soil.1B-Ph1P.2B 0.86667 0.99471 0.871 0.9994 Soil.2B-Ph1P.2B 0.31111 0.99471 0.313 1 Stool.1B-Ph1P.2B -2.24444 0.99471 -2.256 0.5074 Stool.2B-Ph1P.2B -1.33333 0.99471 -1.34 0.9738 Ph1S.2B-Ph1S.1B 2.26667 0.99471 2.279 0.4912 Ph24P.1BPh1S.1B 2.24444 0.99471 2.256 0.5081 Ph24P.2BPh1S.1B 2.26667 0.99471 2.279 0.4915 Ph24S.1BPh1S.1B -1.51111 0.99471 -1.519 0.9357 Ph24S.2BPh1S.1B 0.4 0.99471 0.402 1 Soil.1B-Ph1S.1B 1.84444 0.99471 1.854 0.7875 Soil.2B-Ph1S.1B 1.28889 0.99471 1.296 0.9798 Stool.1B-Ph1S.1B -1.26667 0.99471 -1.273 0.9825 Stool.2B-Ph1S.1B -0.35556 0.99471 -0.357 1 Ph24P.1BPh1S.2B -0.02222 0.99471 -0.022 1 Ph24P.2BPh1S.2B 0 0.99471 0 1 Ph24S.1BPh1S.2B -3.77778 0.99471 -3.798 <0.01 ** Ph24S.2BPh1S.2B -1.86667 0.99471 -1.877 0.7742
Soil.1B-Ph1S.2B -0.42222 0.99471 -0.424 1 Soil.2B-Ph1S.2B -0.97778 0.99471 -0.983 0.9981 Stool.1B-Ph1S.2B -3.53333 0.99471 -3.552 0.02 * Stool.2B-Ph1S.2B -2.62222 0.99471 -2.636 0.259 Ph24P.2BPh24P.1B 0.02222 0.99471 0.022 1 Ph24S.1BPh24P.1B -3.75556 0.99471 -3.776 <0.01 ** Ph24S.2BPh24P.1B -1.84444 0.99471 -1.854 0.7875 Soil.1B-Ph24P.1B -0.4 0.99471 -0.402 1 Soil.2B-Ph24P.1B -0.95556 0.99471 -0.961 0.9984 Stool.1BPh24P.1B -3.51111 0.99471 -3.53 0.0215 * Stool.2BPh24P.1B -2.6 0.99471 -2.614 0.2719 Ph24S.1BPh24P.2B -3.77778 0.99471 -3.798 <0.01 ** Ph24S.2BPh24P.2B -1.86667 0.99471 -1.877 0.7743 Soil.1B-Ph24P.2B -0.42222 0.99471 -0.424 1 Soil.2B-Ph24P.2B -0.97778 0.99471 -0.983 0.9981 Stool.1BPh24P.2B -3.53333 0.99471 -3.552 0.0202 * Stool.2BPh24P.2B -2.62222 0.99471 -2.636 0.2589 Ph24S.2BPh24S.1B 1.91111 0.99471 1.921 0.7458 Soil.1B-Ph24S.1B 3.35556 0.99471 3.373 0.0366 * Soil.2B-Ph24S.1B 2.8 0.99471 2.815 0.1732 Stool.1BPh24S.1B 0.24444 0.99471 0.246 1 Stool.2BPh24S.1B 1.15556 0.99471 1.162 0.9917 Soil.1B-Ph24S.2B 1.44444 0.99471 1.452 0.9529 Soil.2B-Ph24S.2B 0.88889 0.99471 0.894 0.9992 Stool.1BPh24S.2B -1.66667 0.99471 -1.676 0.8797 Stool.2BPh24S.2B -0.75556 0.99471 -0.76 0.9998 Soil.2B-Soil.1B -0.55556 0.99471 -0.559 1 Stool.1B-Soil.1B -3.11111 0.99471 -3.128 0.0762 . Stool.2B-Soil.1B -2.2 0.99471 -2.212 0.5408 Stool.1B-Soil.2B -2.55556 0.99471 -2.569 0.2972 Stool.2B-Soil.2B -1.64444 0.99471 -1.653 0.889 Stool.2B-Stool.1B 0.91111 0.99471 0.916 0.999
Table S8. Post hoc pairwise comparison of significant differences in Shannon diversity of target species derived from different protocols from the 18S rDNA dataset. Protocols Estimate Std. Error Z-value Pr(>|z|) Stool.1B-Ph1S.2B 0.3891827 0.0788487 4.936 <0.01 *** Stool.2B-Ph1S.2B 0.2919968 0.0788487 3.703 0.0117 * Stool.1B-Ph24S.1B 0.3765767 0.0788487 4.776 <0.01 *** Stool.2B-Ph24S.1B 0.2793909 0.0788487 3.543 0.0205 * Stool.1B-Ph24S.2B 0.3221138 0.0788487 4.085 <0.01 ** Figure S1. Comparison of Shannon diversity of target species captured by the different extraction protocols in the 18S rDNA dataset. Table S9. Post hoc pairwise comparison of significant differences in Shannon diversity of target species derived from different protocols from the Crust16S mtDNA dataset. Protocols Estimate Std. Error Z-value Pr(>|z|) Soil.1B-Ph1P.2B 0.41254 0.122292 3.373 0.036 * Soil.1B-Ph24P.1B 0.430272 0.122292 3.518 0.0226 * Soil.1B-Ph24P.2B 0.51634 0.122292 4.222 <0.01 ** Stool.1B-Soil.1B -0.515106 0.122292 -4.212 <0.01 ** Stool.2B-Soil.1B -0.437476 0.122292 -3.577 0.0181 *
Figure S2. Comparison of Shannon diversity of target species captured by the different extraction protocols in the Crust16S mtDNA dataset. Table S10. Post hoc pairwise comparison of significant differences in target species abundance composition derived from different protocols from the 18S rDNA dataset. Observed statistic Free Stepdown Adjusted P-Value Ph24S.1B vs Stool.1B 119.757 0.005 ** Ph24S.1B vs Soil.2B 104.095 0.010 ** Stool.1B vs Soil.2B 102.376 0.010 ** Stool.1B vs Soil.1B 99.982 0.010 ** Ph1P.2B vs Ph24S.1B 97.004 0.010 ** Ph1P.2B vs Soil.2B 92.456 0.015 * Ph1P.2B vs Stool.1B 92.327 0.015 * Stool.2B vs Soil.2B 90.866 0.015 * Ph24S.1B vs Soil.1B 89.635 0.015 * Ph1P.1B vs Stool.1B 88.172 0.015 * Ph1P.2B vs Soil.1B 87.412 0.015 * Stool.2B vs Soil.1B 86.460 0.015 * Ph1P.1B vs Soil.2B 86.076 0.015 * Ph1P.1B vs Ph24S.1B 85.243 0.015 * Ph1S.1B vs Stool.1B 85.224 0.015 * Ph24S.2B vs Stool.1B 80.202 0.020 * Ph1P.1B vs Soil.1B 77.748 0.030 * Ph24P.1B vs Stool.1B 74.187 0.035 * Ph1P.2B vs Ph24P.1B 73.204 0.035 * Ph24P.2B vs Ph24S.1B 73.140 0.035 * Ph24P.2B vs Stool.2B 72.983 0.035 * Ph24P.2B vs Stool.1B 72.282 0.035 * Ph1P.1B vs Stool.2B 70.222 0.045 *
Ph24S.2B-Ph1S.2B 0.28889 1.1778 0.245 1 Soil.1B-Ph1S.2B 5.15556 1.1778 4.377 <0.01 *** Soil.2B-Ph1S.2B 6.15556 1.1778 5.226 <0.01 *** Stool.1B-Ph1S.2B 5.93333 1.1778 5.038 <0.01 *** Stool.2B-Ph1S.2B 2.95556 1.1778 2.509 0.3331 Ph24P.2BPh24P.1B -1.8 1.1778 -1.528 0.9332 Ph24S.1BPh24P.1B -4.51111 1.1778 -3.83 <0.01 ** Ph24S.2BPh24P.1B -3.42222 1.1778 -2.906 0.1383 Soil.1B-Ph24P.1B 1.44444 1.1778 1.226 0.987 Soil.2B-Ph24P.1B 2.44444 1.1778 2.075 0.6406 Stool.1B-Ph24P.1B 2.22222 1.1778 1.887 0.7678 Stool.2B-Ph24P.1B -0.75556 1.1778 -0.641 1 Ph24S.1BPh24P.2B -2.71111 1.1778 -2.302 0.4753 Ph24S.2BPh24P.2B -1.62222 1.1778 -1.377 0.9679 Soil.1B-Ph24P.2B 3.24444 1.1778 2.755 0.2004 Soil.2B-Ph24P.2B 4.24444 1.1778 3.604 0.0163 * Stool.1B-Ph24P.2B 4.02222 1.1778 3.415 0.0314 * Stool.2B-Ph24P.2B 1.04444 1.1778 0.887 0.9992 Ph24S.2BPh24S.1B 1.08889 1.1778 0.925 0.9989 Soil.1B-Ph24S.1B 5.95556 1.1778 5.056 <0.01 *** Soil.2B-Ph24S.1B 6.95556 1.1778 5.906 <0.01 *** Stool.1B-Ph24S.1B 6.73333 1.1778 5.717 <0.01 *** Stool.2B-Ph24S.1B 3.75556 1.1778 3.189 0.0641 . Soil.1B-Ph24S.2B 4.86667 1.1778 4.132 <0.01 ** Soil.2B-Ph24S.2B 5.86667 1.1778 4.981 <0.01 *** Stool.1B-Ph24S.2B 5.64444 1.1778 4.792 <0.01 *** Stool.2B-Ph24S.2B 2.66667 1.1778 2.264 0.5025 Soil.2B-Soil.1B 1 1.1778 0.849 0.9995 Stool.1B-Soil.1B 0.77778 1.1778 0.66 1 Stool.2B-Soil.1B -2.2 1.1778 -1.868 0.779 Stool.1B-Soil.2B -0.22222 1.1778 -0.189 1 Stool.2B-Soil.2B -3.2 1.1778 -2.717 0.2184 Stool.2B-Stool.1B -2.97778 1.1778 -2.528 0.3197 Table S16. Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw 18S rDNA dataset. Protocols Estimate Std Error z value Pr(>|z|) Ph1P.2B-Ph1P.1B 0.0104359 0.0926887 0.113 1 Ph1S.1B-Ph1P.1B 0.2779648 0.0926887 2.999 0.1096
Ph1S.2B-Ph1P.1B 0.1382541 0.0926887 1.492 0.9431 Ph24P.1B-Ph1P.1B - 0.2363032 0.0926887 -2.549 0.3091 Ph24P.2B-Ph1P.1B - 0.2632176 0.0926887 -2.84 0.1627 Ph24S.1B-Ph1P.1B 0.0810135 0.0926887 0.874 0.9993 Ph24S.2B-Ph1P.1B - 0.1262406 0.0926887 -1.362 0.9707 Soil.1B-Ph1P.1B - 0.2181565 0.0926887 -2.354 0.4382 Soil.2B-Ph1P.1B -0.21896 0.0926887 -2.362 0.4331 Stool.1B-Ph1P.1B 0.0768936 0.0926887 0.83 0.9996 Stool.2B-Ph1P.1B - 0.0630501 0.0926887 -0.68 0.9999 Ph1S.1B-Ph1P.2B 0.2675289 0.0926887 2.886 0.1463 Ph1S.2B-Ph1P.2B 0.1278182 0.0926887 1.379 0.9676 Ph24P.1B-Ph1P.2B - 0.2467391 0.0926887 -2.662 0.2458 Ph24P.2B-Ph1P.2B - 0.2736535 0.0926887 -2.952 0.1235 Ph24S.1B-Ph1P.2B 0.0705776 0.0926887 0.761 0.9998 Ph24S.2B-Ph1P.2B - 0.1366765 0.0926887 -1.475 0.9477 Soil.1B-Ph1P.2B - 0.2285924 0.0926887 -2.466 0.3611 Soil.2B-Ph1P.2B - 0.2293959 0.0926887 -2.475 0.3557 Stool.1B-Ph1P.2B 0.0664577 0.0926887 0.717 0.9999 Stool.2B-Ph1P.2B -0.073486 0.0926887 -0.793 0.9997 Ph1S.2B-Ph1S.1B - 0.1397108 0.0926887 -1.507 0.9391 Ph24P.1B-Ph1S.1B -0.514268 0.0926887 -5.548 <0.01 *** Ph24P.2B-Ph1S.1B - 0.5411825 0.0926887 -5.839 <0.01 *** Ph24S.1B-Ph1S.1B - 0.1969513 0.0926887 -2.125 0.6044 Ph24S.2B-Ph1S.1B - 0.4042054 0.0926887 -4.361 <0.01 *** Soil.1B-Ph1S.1B - 0.4961214 0.0926887 -5.353 <0.01 *** Soil.2B-Ph1S.1B - 0.4969248 0.0926887 -5.361 <0.01 *** Stool.1B-Ph1S.1B - 0.2010713 0.0926887 -2.169 0.5723 Stool.2B-Ph1S.1B - 0.3410149 0.0926887 -3.679 0.0126 * Ph24P.1B-Ph1S.2B - 0.3745572 0.0926887 -4.041 <0.01 **
Ph24P.2B-Ph1S.2B - 0.4014717 0.0926887 -4.331 <0.01 ** Ph24S.1B-Ph1S.2B - 0.0572406 0.0926887 -0.618 1 Ph24S.2B-Ph1S.2B - 0.2644946 0.0926887 -2.854 0.1581 Soil.1B-Ph1S.2B - 0.3564106 0.0926887 -3.845 <0.01 ** Soil.2B-Ph1S.2B - 0.3572141 0.0926887 -3.854 <0.01 ** Stool.1B-Ph1S.2B - 0.0613605 0.0926887 -0.662 1 Stool.2B-Ph1S.2B - 0.2013042 0.0926887 -2.172 0.5706 Ph24P.2BPh24P.1B - 0.0269144 0.0926887 -0.29 1 Ph24S.1BPh24P.1B 0.3173167 0.0926887 3.423 0.0304 * Ph24S.2BPh24P.1B 0.1100626 0.0926887 1.187 0.99 Soil.1B-Ph24P.1B 0.0181467 0.0926887 0.196 1 Soil.2B-Ph24P.1B 0.0173432 0.0926887 0.187 1 Stool.1B-Ph24P.1B 0.3131968 0.0926887 3.379 0.0352 * Stool.2B-Ph24P.1B 0.1732531 0.0926887 1.869 0.7785 Ph24S.1BPh24P.2B 0.3442311 0.0926887 3.714 0.011 * Ph24S.2BPh24P.2B 0.136977 0.0926887 1.478 0.9469 Soil.1B-Ph24P.2B 0.0450611 0.0926887 0.486 1 Soil.2B-Ph24P.2B 0.0442576 0.0926887 0.477 1 Stool.1B-Ph24P.2B 0.3401112 0.0926887 3.669 0.0128 * Stool.2B-Ph24P.2B 0.2001675 0.0926887 2.16 0.5791 Ph24S.2BPh24S.1B - 0.2072541 0.0926887 -2.236 0.5238 Soil.1B-Ph24S.1B -0.29917 0.0926887 -3.228 0.0571 . Soil.2B-Ph24S.1B - 0.2999735 0.0926887 -3.236 0.0552 . Stool.1B-Ph24S.1B - 0.0041199 0.0926887 -0.044 1 Stool.2B-Ph24S.1B - 0.1440636 0.0926887 -1.554 0.9251 Soil.1B-Ph24S.2B - 0.0919159 0.0926887 -0.992 0.9979 Soil.2B-Ph24S.2B - 0.0927194 0.0926887 -1 0.9977 Stool.1B-Ph24S.2B 0.2031342 0.0926887 2.192 0.5552 Stool.2B-Ph24S.2B 0.0631905 0.0926887 0.682 0.9999
Soil.2B-Soil.1B - 0.0008035 0.0926887 -0.009 1 Stool.1B-Soil.1B 0.2950501 0.0926887 3.183 0.0648 . Stool.2B-Soil.1B 0.1551064 0.0926887 1.673 0.8805 Stool.1B-Soil.2B 0.2958536 0.0926887 3.192 0.0638 . Stool.2B-Soil.2B 0.1559099 0.0926887 1.682 0.8766 Stool.2B-Stool.1B - 0.1399437 0.0926887 -1.51 0.9383 Table S17. Summary of zOTU richness captured by each extraction protocol for the raw Crust16S mtDNA dataset. Extraction protocol Mean zOTU richness SD Ph1P.1B 10.5 3.41 Ph1P.2B 7.71 4.66 Ph1S.1B 8.18 2.94 Ph1S.2B 8.58 3.91 Ph24P.1B 9.18 3.46 Ph24P.2B 9.98 3.15 Ph24S.1B 6.36 2.9 Ph24S.2B 8.64 2.18 Soil.1B 8.44 3.64 Soil.2B 8.56 4.7 Stool.1B 4.51 3.74 Stool.2B 7.02 3.08 Table S18. Post hoc pairwise comparison of differences in zOTU richness derived from different protocols from the raw Crust16S mtDNA dataset. Protocols Estimate Std Error z value Pr(>|z|) Ph1P.2B-Ph1P.1B -2.75556 0.72663 -3.792 <0.01 ** Ph1S.1B-Ph1P.1B -2.28889 0.72663 -3.15 0.0703 . Ph1S.2B-Ph1P.1B -1.88889 0.72663 -2.6 0.2793 Ph24P.1B-Ph1P.1B -1.28889 0.72663 -1.774 0.8323 Ph24P.2B-Ph1P.1B -0.48889 0.72663 -0.673 0.9999 Ph24S.1B-Ph1P.1B -4.11111 0.72663 -5.658 <0.01 *** Ph24S.2B-Ph1P.1B -1.82222 0.72663 -2.508 0.3337 Soil.1B-Ph1P.1B -2.02222 0.72663 -2.783 0.1857 Soil.2B-Ph1P.1B -1.91111 0.72663 -2.63 0.2627 Stool.1B-Ph1P.1B -5.95556 0.72663 -8.196 <0.01 *** Stool.2B-Ph1P.1B -3.44444 0.72663 -4.74 <0.01 *** Ph1S.1B-Ph1P.2B 0.46667 0.72663 0.642 1 Ph1S.2B-Ph1P.2B 0.86667 0.72663 1.193 0.9897 Ph24P.1B-Ph1P.2B 1.46667 0.72663 2.018 0.6809
Ph24P.2B-Ph1P.2B 2.26667 0.72663 3.119 0.0769 . Ph24S.1B-Ph1P.2B -1.35556 0.72663 -1.866 0.781 Ph24S.2B-Ph1P.2B 0.93333 0.72663 1.284 0.9813 Soil.1B-Ph1P.2B 0.73333 0.72663 1.009 0.9975 Soil.2B-Ph1P.2B 0.84444 0.72663 1.162 0.9916 Stool.1B-Ph1P.2B -3.2 0.72663 -4.404 <0.01 *** Stool.2B-Ph1P.2B -0.68889 0.72663 -0.948 0.9986 Ph1S.2B-Ph1S.1B 0.4 0.72663 0.55 1 Ph24P.1B-Ph1S.1B 1 0.72663 1.376 0.9681 Ph24P.2B-Ph1S.1B 1.8 0.72663 2.477 0.3544 Ph24S.1B-Ph1S.1B -1.82222 0.72663 -2.508 0.3345 Ph24S.2B-Ph1S.1B 0.46667 0.72663 0.642 1 Soil.1B-Ph1S.1B 0.26667 0.72663 0.367 1 Soil.2B-Ph1S.1B 0.37778 0.72663 0.52 1 Stool.1B-Ph1S.1B -3.66667 0.72663 -5.046 <0.01 *** Stool.2B-Ph1S.1B -1.15556 0.72663 -1.59 0.913 Ph24P.1B-Ph1S.2B 0.6 0.72663 0.826 0.9996 Ph24P.2B-Ph1S.2B 1.4 0.72663 1.927 0.7428 Ph24S.1B-Ph1S.2B -2.22222 0.72663 -3.058 0.0926 . Ph24S.2B-Ph1S.2B 0.06667 0.72663 0.092 1 Soil.1B-Ph1S.2B -0.13333 0.72663 -0.183 1 Soil.2B-Ph1S.2B -0.02222 0.72663 -0.031 1 Stool.1B-Ph1S.2B -4.06667 0.72663 -5.597 <0.01 *** Stool.2B-Ph1S.2B -1.55556 0.72663 -2.141 0.5932 Ph24P.2BPh24P.1B 0.8 0.72663 1.101 0.9948 Ph24S.1BPh24P.1B -2.82222 0.72663 -3.884 <0.01 ** Ph24S.2BPh24P.1B -0.53333 0.72663 -0.734 0.9999 Soil.1B-Ph24P.1B -0.73333 0.72663 -1.009 0.9975 Soil.2B-Ph24P.1B -0.62222 0.72663 -0.856 0.9995 Stool.1B-Ph24P.1B -4.66667 0.72663 -6.422 <0.01 *** Stool.2B-Ph24P.1B -2.15556 0.72663 -2.967 0.1184 Ph24S.1BPh24P.2B -3.62222 0.72663 -4.985 <0.01 *** Ph24S.2BPh24P.2B -1.33333 0.72663 -1.835 0.7989 Soil.1B-Ph24P.2B -1.53333 0.72663 -2.11 0.6165 Soil.2B-Ph24P.2B -1.42222 0.72663 -1.957 0.7231 Stool.1B-Ph24P.2B -5.46667 0.72663 -7.523 <0.01 *** Stool.2B-Ph24P.2B -2.95556 0.72663 -4.067 <0.01 ** Ph24S.2BPh24S.1B 2.28889 0.72663 3.15 0.071 . Soil.1B-Ph24S.1B 2.08889 0.72663 2.875 0.1493
Soil.2B-Ph24S.1B 2.2 0.72663 3.028 0.1009 Stool.1B-Ph24S.1B -1.84444 0.72663 -2.538 0.3155 Stool.2B-Ph24S.1B 0.66667 0.72663 0.917 0.999 Soil.1B-Ph24S.2B -0.2 0.72663 -0.275 1 Soil.2B-Ph24S.2B -0.08889 0.72663 -0.122 1 Stool.1B-Ph24S.2B -4.13333 0.72663 -5.688 <0.01 *** Stool.2B-Ph24S.2B -1.62222 0.72663 -2.233 0.5256 Soil.2B-Soil.1B 0.11111 0.72663 0.153 1 Stool.1B-Soil.1B -3.93333 0.72663 -5.413 <0.01 *** Stool.2B-Soil.1B -1.42222 0.72663 -1.957 0.7224 Stool.1B-Soil.2B -4.04444 0.72663 -5.566 <0.01 *** Stool.2B-Soil.2B -1.53333 0.72663 -2.11 0.6153 Stool.2B-Stool.1B 2.51111 0.72663 3.456 0.0269 * Table S19. Post hoc pairwise comparison of differences in Shannon diversity derived from different protocols from the raw Crust16S mtDNA dataset. Protocols Estimate Std Error z value Pr(>|z|) Ph1P.2B-Ph1P.1B - 0.1513174 0.107553 -1.407 0.962 Ph1S.1B-Ph1P.1B 0.1113204 0.107553 1.035 0.997 Ph1S.2B-Ph1P.1B 0.0909475 0.107553 0.846 1 Ph24P.1B-Ph1P.1B 0.0898855 0.107553 0.836 1 Ph24P.2B-Ph1P.1B 0.010241 0.107553 0.095 1 Ph24S.1B-Ph1P.1B 0.1200061 0.107553 1.116 0.994 Ph24S.2B-Ph1P.1B 0.1108741 0.107553 1.031 0.997 Soil.1B-Ph1P.1B 0.1522184 0.107553 1.415 0.961 Soil.2B-Ph1P.1B -0.048796 0.107553 -0.454 1 Stool.1B-Ph1P.1B - 0.1529458 0.107553 -1.422 0.959 Stool.2B-Ph1P.1B 0.048904 0.107553 0.455 1 Ph1S.1B-Ph1P.2B 0.2626378 0.107553 2.442 0.377 Ph1S.2B-Ph1P.2B 0.242265 0.107553 2.253 0.511 Ph24P.1B-Ph1P.2B 0.241203 0.107553 2.243 0.518 Ph24P.2B-Ph1P.2B 0.1615584 0.107553 1.502 0.94 Ph24S.1B-Ph1P.2B 0.2713235 0.107553 2.523 0.326 Ph24S.2B-Ph1P.2B 0.2621916 0.107553 2.438 0.381 Soil.1B-Ph1P.2B 0.3035358 0.107553 2.822 0.17 Soil.2B-Ph1P.2B 0.1025215 0.107553 0.953 0.999 Stool.1B-Ph1P.2B - 0.0016283 0.107553 -0.015 1 Stool.2B-Ph1P.2B 0.2002215 0.107553 1.862 0.782 Ph1S.2B-Ph1S.1B - 0.0203729 0.107553 -0.189 1 Ph24P.1B-Ph1S.1B - 0.0214349 0.107553 -0.199 1
Ph24P.2B-Ph1S.1B - 0.1010794 0.107553 -0.94 0.999 Ph24S.1B-Ph1S.1B 0.0086857 0.107553 0.081 1 Ph24S.2B-Ph1S.1B - 0.0004463 0.107553 -0.004 1 Soil.1B-Ph1S.1B 0.040898 0.107553 0.38 1 Soil.2B-Ph1S.1B - 0.1601164 0.107553 -1.489 0.944 Stool.1B-Ph1S.1B - 0.2642662 0.107553 -2.457 0.367 Stool.2B-Ph1S.1B - 0.0624164 0.107553 -0.58 1 Ph24P.1B-Ph1S.2B -0.001062 0.107553 -0.01 1 Ph24P.2B-Ph1S.2B - 0.0807065 0.107553 -0.75 1 Ph24S.1B-Ph1S.2B 0.0290585 0.107553 0.27 1 Ph24S.2B-Ph1S.2B 0.0199266 0.107553 0.185 1 Soil.1B-Ph1S.2B 0.0612708 0.107553 0.57 1 Soil.2B-Ph1S.2B - 0.1397435 0.107553 -1.299 0.979 Stool.1B-Ph1S.2B - 0.2438933 0.107553 -2.268 0.5 Stool.2B-Ph1S.2B - 0.0420435 0.107553 -0.391 1 Ph24P.2BPh24P.1B - 0.0796445 0.107553 -0.741 1 Ph24S.1BPh24P.1B 0.0301206 0.107553 0.28 1 Ph24S.2BPh24P.1B 0.0209886 0.107553 0.195 1 Soil.1B-Ph24P.1B 0.0623328 0.107553 0.58 1 Soil.2B-Ph24P.1B - 0.1386815 0.107553 -1.289 0.981 Stool.1B-Ph24P.1B - 0.2428313 0.107553 -2.258 0.508 Stool.2B-Ph24P.1B - 0.0409815 0.107553 -0.381 1 Ph24S.1BPh24P.2B 0.1097651 0.107553 1.021 0.997 Ph24S.2BPh24P.2B 0.1006331 0.107553 0.936 0.999 Soil.1B-Ph24P.2B 0.1419774 0.107553 1.32 0.977 Soil.2B-Ph24P.2B -0.059037 0.107553 -0.549 1 Stool.1B-Ph24P.2B - 0.1631868 0.107553 -1.517 0.936 Stool.2B-Ph24P.2B 0.038663 0.107553 0.359 1 Ph24S.2BPh24S.1B -0.009132 0.107553 -0.085 1
Soil.1B-Ph24S.1B 0.0322123 0.107553 0.3 1 Soil.2B-Ph24S.1B -0.168802 0.107553 -1.569 0.92 Stool.1B-Ph24S.1B - 0.2729518 0.107553 -2.538 0.316 Stool.2B-Ph24S.1B - 0.0711021 0.107553 -0.661 1 Soil.1B-Ph24S.2B 0.0413442 0.107553 0.384 1 Soil.2B-Ph24S.2B - 0.1596701 0.107553 -1.485 0.945 Stool.1B-Ph24S.2B - 0.2638199 0.107553 -2.453 0.37 Stool.2B-Ph24S.2B - 0.0619701 0.107553 -0.576 1 Soil.2B-Soil.1B - 0.2010143 0.107553 -1.869 0.779 Stool.1B-Soil.1B - 0.3051641 0.107553 -2.837 0.164 Stool.2B-Soil.1B - 0.1033143 0.107553 -0.961 0.998 Stool.1B-Soil.2B - 0.1041498 0.107553 -0.968 0.998 Stool.2B-Soil.2B 0.0977 0.107553 0.908 0.999 Stool.2B-Stool.1B 0.2018498 0.107553 1.877 0.774 Taxonomic composition For both 18S rDNA and Crust16S mtDNA there was a significant effect of extraction protocol on composition based on both abundance and presence-absence (P < 0.001 in all cases). Pairwise protocol comparisons may be found below after the figure.
Figure S7. Relative abundance of (a) unique classes detected in the raw 18S rDNA dataset and (b) unique species detected in the raw Crust16S mtDNA dataset. Table S20. Post hoc pairwise comparison of significant differences in species abundance compositional data derived from different protocols from the raw 18S rDNA dataset. Protocols Observed statistic Adjusted P-value Soil.1B vs Stool.1B 1147.8 0.005 ** Soil.2B vs Stool.1B 1098.2 0.005 ** Ph1P.2B vs Ph24S.1B 938.4 0.005 ** Soil.1B vs Stool.2B 931.9 0.005 ** Ph1P.1B vs Stool.1B 930.5 0.005 ** Ph1S.1B vs Stool.1B 921.4 0.005 ** Ph1P.2B vs Stool.1B 914.2 0.005 ** Ph24S.1B vs Stool.1B 911.9 0.005 ** Soil.2B vs Stool.2B 907.5 0.005 ** Ph24P.1B vs Stool.1B 893.5 0.005 ** Ph1P.1B vs Ph24S.1B 847.6 0.005 ** Ph1P.2B vs Ph1S.1B 835 0.005 ** Ph24S.1B vs Soil.2B 828.3 0.005 ** Ph1S.1B vs Soil.1B 810 0.005 ** Ph24P.2B vs Stool.1B 809.9 0.005 ** Ph1S.1B vs Soil.2B 802.6 0.005 ** Ph24S.1B vs Soil.1B 799.3 0.005 ** Ph1P.1B vs Stool.2B 781.1 0.005 ** Ph1S.2B vs Stool.1B 776.6 0.005 ** Ph24S.2B vs Soil.1B 774.2 0.005 ** Ph1P.1B vs Ph1S.1B 771.2 0.005 ** Ph1P.2B vs Stool.2B 770.4 0.005 **
Ph24S.2B vs Stool.1B 769 0.005 ** Ph24S.2B vs Soil.2B 766.1 0.005 ** Ph24P.1B vs Stool.2B 733.8 0.005 ** Ph1S.1B vs Ph24P.1B 720 0.005 ** Ph1P.2B vs Ph24S.2B 718.7 0.005 ** Ph24P.1B vs Ph24S.1B 713.5 0.005 ** Ph1S.1B vs Ph24P.2B 710.2 0.005 ** Ph24P.2B vs Ph24S.1B 707.4 0.005 ** Ph24P.2B vs Stool.2B 688.5 0.005 ** Ph1S.1B vs Stool.2B 684.9 0.005 ** Ph1S.2B vs Soil.1B 682.8 0.005 ** Ph24S.1B vs Stool.2B 676.4 0.005 ** Ph1S.2B vs Soil.2B 660.3 0.005 ** Ph1P.1B vs Ph24S.2B 654.4 0.005 ** Ph1P.2B vs Ph1S.2B 647.5 0.005 ** Ph24P.1B vs Ph24S.2B 618.5 0.005 ** Ph24P.2B vs Ph24S.2B 580.3 0.005 ** Ph1S.2B vs Ph24P.1B 574.9 0.005 ** Ph1S.2B vs Stool.2B 572.5 0.005 ** Ph24S.2B vs Stool.2B 567.7 0.005 ** Ph1P.1B vs Ph1S.2B 552.4 0.005 ** Ph1P.2B vs Soil.2B 537 0.005 ** Ph1S.2B vs Ph24P.2B 512.4 0.005 ** Ph1P.1B vs Soil.2B 502.7 0.005 ** Ph1P.2B vs Soil.1B 485.9 0.005 ** Ph1P.1B vs Soil.1B 473.8 0.005 ** Ph24S.1B vs Ph24S.2B 459.4 0.005 ** Ph24P.2B vs Soil.1B 439.9 0.005 ** Ph24P.2B vs Soil.2B 433 0.005 ** Ph1S.2B vs Ph24S.1B 414.7 0.005 ** Ph1S.1B vs Ph24S.1B 401.3 0.005 ** Ph24P.1B vs Soil.2B 398.9 0.005 ** Ph24P.1B vs Soil.1B 389.2 0.01 ** Ph1P.2B vs Ph24P.1B 384.1 0.01 ** Ph1S.1B vs Ph1S.2B 370 0.015 * Ph1S.1B vs Ph24S.2B 369.3 0.015 * Ph1P.1B vs Ph24P.1B 339.8 0.03 * Ph1P.2B vs Ph24P.2B 337.9 0.03 * Ph1S.2B vs Ph24S.2B 324.3 0.05 *