SUPPLEMENTAL MATERIALS AND METHODS Construction of
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1 SUPPLEMENTAL MATERIALS AND METHODS 2 Construction of reference and OTU phylogenies. A bacterial phylogeny suitable for use with 3 the program pplacer was constructed by Kembel et al. (2012) using 16S sequences from the 4 Greengenes database (DeSantis et al., 2006), but no such phylogeny was available for fungi. To 5 construct the reference phylogeny for fungi, full and nearly full-length fungal LSU sequences 6 from the Assembling the Fungal Tree of Life Project (Celio et al., 2007) were downloaded from 7 the GenBank nucleotide database, aligned using MAFFT version 7.0 (Katoh & Standley, 2013) 8 and then filtered to remove any position with > 15% gaps or variable bases in mothur (Schloss 9 et al., 2009). A maximum-likelihood phylogeny was built using RAxML v 7.2.7 (Stamatakis et al., 10 2005), with the GTR + gamma + I model. To place environmental OTUs onto their respective 11 reference phylogenies (maximum likelihood fungal LSU or bacterial 16S from Kembel et al. 12 (2012), representative fungal and bacterial OTUs were aligned to their respective reference tree 13 databases using PyNAST (Caporaso et al., 2010), with a BLAST cutoff of 0.6. Bacterial OTU 14 sequences were masked using the Greengenes file from QIIME or the fungal LSU mask file 15 generated during filtering of the AFTOL sequences as described above. Filtered fungal and 16 bacterial OTU sequences were placed onto their respective reference trees using pplacer 17 (Matsen et al., 2010). To visualize the distribution of active and dormant OTUs across the 18 bacterial and fungal OTU phylogenies, we used the Interactive Tree of Life (itol.embl.de) to 19 color branches based on classification of OTUs to the phylum level from the Ribosomal 20 Database Project. OTUs classified with a bootstrap value less than 50% were colored in grey. An 21 outer ring was then added to indicate the inferred state of each OTU based on the ratio of rRNA 22 to rDNA (active, dormant, unknown; Supplemental Fig. 1A, 1B). 1 23 Null model trials for patterns of phylogenetic clustering. We used two additional null models 24 to determine of the patterns of phylogenetic clustering between active and dormant 25 communities were robust. Using the ses.mpd script in the R package picante (Kembel et al., 26 2010) we selected a null model which randomized all taxa across the tips of the phylogeny 27 (“taxa.labels in picante), and the independent swap model (“independentswap” in picante) 28 based on the algorithm by Gotelli (2000). Each null model was run for 99 trials, using 29 rarefaction to randomly sample the bacterial and fungal communities to their lowest sequence 30 numbers (400 and 1000, respectively). Statistical analyses were conducted as described for the 31 “frequency” null model described in the main text. 32 33 SUPPLEMENTAL RESULTS 34 Null models. Paired t-tests showed significant differences between the active and dormant 35 bacterial communities using both the “independentswap” model (IS; t = 5.57, p < 0.001, DF = 8) 36 and the “taxa.labels” model (TL; t = 3.82, p = 0.005). Similar patterns were found when 37 comparing the rRNA versus the rDNA for these same models (IS: t = 10.84, p < 0.001, TL: 5.36, p 38 < 0.001). We also found significant differences for fungal communities, with significantly higher 39 clustering for the dormant and rDNA communities compared to the active and rRNA 40 communities, respectively, the opposite of the pattern observed for bacteria. Significant 41 differences were found between the active and dormant fungal community using the 42 “independentswap” model (t = -11.8, p < 0.001, DF = 11) and the “taxa.labels” model (t = -4.01, 43 p = 0.002), and these patterns held when the rRNA versus the rDNA communities were 44 compared (IS: t = -4.74, p < 0.001, TL: t = -6.94, p < 0.001). 2 45 Regression analysis using the additional null models showed significant correlations for 46 the active bacterial communities for both pH (IS: F = 11.31, R2 = 0.56, p = 0.01, DF = 1,7, TL: F = 47 33.1, R2 = 0.80, p < 0.001) and for carbon to nitrogen ratio (IS: F = 6.42, R2 = 0.40, p = 0.04, TL: F 48 = 32.9, R2 = 0.80, p < 0.001). No correlations were found for either model with the dormant 49 bacterial community (all p > 0.05). Analysis of the fungal communities likewise showed no 50 significant correlations between any community (active or dormant) and either pH or carbon to 51 nitrogen ratio, regardless of the null model used (all p > 0.05). 52 53 References 54 Caporaso JG, Bittinger K, Bushman FD, DeSantis TZ, Andersen GL, Knight R. (2010). PyNAST: a 55 flexible tool for aligning sequences to a template alignment. Bioinformatics 26:266–267. 56 57 Celio GJ, Padamsee M, Dentinger BTM, Bauer R, McLaughlin DJ. (2007). Assembling the Fungal Tree of Life: constructing the Structural and Biochemical Database. Mycologia 98:850–859. 58 DeSantis TZ, Hugenholtz P, Larsen N, Rojas M, Brodie EL, Keller K, et al. (2006). Greengenes, a 59 chimera-checked 16S rRNA gene database and workbench compatible with ARB. Applied 60 and Environmental Microbiology 72:5069–5072. 61 Gotelli NJ. (2000). Null model analysis of species co-occurrence patterns. Ecology 81:2606– 62 2621. http://www.esajournals.org/doi/abs/10.1890/0012- 63 9658%282000%29081%5B2606%3ANMAOSC%5D2.0.CO%3B2. 64 65 66 Katoh K, Standley DM. (2013). MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 30:772–780. Kembel SW, Cowan PD, Helmus MR, Cornwell WK, Morlon H, Ackerly DD, et al. (2010). Picante: 3 67 R tools for integrating phylogenies and ecology. Bioinformatics 26:1463–1464. 68 Kembel SW, Wu M, Eisen JA, Green JL. (2012). Incorporating 16S gene copy number information 69 improves estimates of microbial diversity and abundance. Kembel, SW, Wu, M, Eisen, JA, & 70 Green, JL (eds). PLoS Comput Biol 8:e1002743. 71 http://dx.plos.org/10.1371/journal.pcbi.1002743.s004. 72 Matsen FA, Kodner RB, Armbrust EV. (2010). pplacer: linear time maximum-likelihood and 73 Bayesian phylogenetic placement of sequences onto a fixed reference tree. BMC 74 Bioinformatics 11:538. 75 Schloss PD, Westcott SL, Ryabin T, Hall JR, Hartmann M, Hollister EB, et al. (2009). Introducing 76 mothur: open-source, platform-independent, community-supported software for 77 describing and comparing microbial communities. Applied and Environmental Microbiology 78 75:7537–7541. 79 Stamatakis A, Ludwig T, Meier H. (2005). RAxML-II: a program for sequential, parallel and 80 distributed inference of large phylogenetic trees. Concurrency Computat: Pract Exper 81 17:1705–1723. 82 83 SUPPLEMENTAL FIGURE LEGENDS 84 Supplemental Fig. 1. Phylogenies of environmental OTUs for bacteria, A and fungi B using 85 placement of representative OTUs onto reference phylogenies. The outer ring indicates 86 classification of each OTU as either active (green), dormant (blue), or undetermined (red). 87 Branch colors represent classification of OTUs to the phylum level using the RDP 16S bacterial 88 or the LSU fungi databases. Branches of any OTU that was classified with a bootstrap value 4 89 below 50% were colored in grey. 90 Supplemental Fig 2. Mean phylogenetic clustering using the “independentswap” null model for 91 bacterial communities, A and fungal communities, B and the “taxa.labels” null model for 92 bacterial communities, C and fungal communities, D showed similar patterns to the 93 “frequency” null model, although whether samples were significantly different from random 94 using permutation tests varied depending upon which null model was used. As patterns were 95 similar for active versus dormant and rRNA versus rDNA, only activity based comparisons are 96 shown for clarity. Asterisks indicate significant differences based on paired t-tests. 97 Supplemental Fig. 3. Regression analysis of active (black symbols) and dormant (grey symbols) 98 communities using the “independentswap” null model (triangles) or the “taxa.labels” null 99 model (squares). Significant correlations were found for the active bacterial communities across 100 gradients of pH, A and carbon to nitrogen ratio, C, with no corresponding correlations found for 101 the dormant community. No significant correlations were found for either the active or the 102 dormant fungal communities across the pH, B or the carbon to nitrogen ratio, D environmental 103 gradients. 104 105 5 SUPPLEMENTAL FIGURES 107 Fig. 1A otu5516 otu7346 otu550 otu7822 otu5842 otu3839 otu6125 otu1749 otu6471 otu670 otu3977 otu3712 otu7412 otu3562 otu7434 otu1117 otu6147 otu4981 otu5727 otu333 otu927 otu6474 otu3930 otu7933 otu1934 otu1080 otu4286 otu6225 otu730 otu7334 otu8285 otu523 otu6357 otu6223 otu4905 otu898 otu3233 otu984 otu991 otu1353 otu1375 otu3832 otu1030 otu2639 otu7406 otu4250 otu5293 otu6645 otu4548 otu6283 otu929 otu8121 otu4790 7 otu993 3 otu294 otu752 otu886 otu624 4 otu79 5 otu150 otu780 8 otu293 6 otu413 9 otu170 otu145 7 otu716 1 otu675 51 otu105 26 otu43 00 otu52 93 otu37 37 otu36 71 otu13 38 3 otu42 otu36 67 otu53 3 otu80 51 otu26 32 otu39 49 39 otu37 45 otu82 50 otu43 55 otu76 31 otu82 3 otu26 18 otu39 otu88 20 11 otu39 2 otu80 5 otu53 otu98 00 otu91 19 39 otu30 otu60 094 otu13 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u4 23 u9 ot 30 24 u7 ot 104 66 u6 07 ot 06 u6 917 82 u7 ot 99 u5 23 u2 35 u1 ot 85 84 u3 75 u8 ot 95 u4 37 ot u5 537 01 u6 25 ot u5 u1 11 ot u5 34 u6 44 7295 ot u5 42 u7 39 ot u1 29 u2 52 ot u5 93 530 u6 50 ot 48 u4 79 u3 ot 52 894 u6 66 u1 u3 86 oot u6 39 u3 3010 97 ot tu u4 38 u7 50 58 00 3636 uu1 14 80 ooot u7 36 ot tu5 u6 80 u6 33 4162 54 ttu 535 ootu 779 452 uu44 56922 ooot ttuuu6 4086091 72 oottu1 18615 208264003 ootttuuu187195 1 o 0 2 1 138 ooottuu65761627351647 ootttuu23737590935741 oottuuu46517820841948602 oootttuu4536635291155727 oo tt uu87624922390242 oo ttuu7348519428646 oo ttuu3532734391135920 ootttuuu5236217484172054 oo tt uu 347288483871 oo ttuu7821375689251969 oo tttuu713221403414669 oo tt uuu13751936826637 oootttuu4576411818821276282 ooottt uu2312286012564 oottt uuu24616013256402512763 ooottttuuuu32347162099079705825028640 o oo t u 4 55 0 8 oo ttt uu 241114791391723 ooo tt uuu531134941172293 ooo tttuuu 2776551 04 418 oooottttuuu8374270768303725748 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otu425 otu180 otu920 otu293 otu437 otu56 7 otu1114 otu1483 otu1024 otu2190 otu1056 otu811 otu1061 otu2348 otu1275 otu1258 otu1011 otu275 otu858 otu1043 otu1152 otu380 otu226 otu2145 otu1594 otu1451 otu35 otu318 otu968 otu1579 otu1390 otu2014 otu213 otu442 otu1333 otu432 otu82 otu431 otu796 otu283 otu816 otu1915 otu531 otu1836 otu1092 otu1596 otu460 otu388 otu2242 otu1278 otu1670 otu1735 otu1575 otu1748 otu2053 otu524 otu2188 otu483 otu2227 otu272 otu2184 otu1130 otu1000 otu454 otu565 otu4911 otu102 1610 otu otu939 6 otu150 otu132 otu2381 3 otu130 otu200 otu6638 otu198 2080 otu otu92 otu462 otu600 otu109 0 otu189 11444 otuu15 ot u3364 ot 40 otuu4655 otu104 ototu486 34 74 otu6 otu31104 50 2 otu2 05 3 otu1 1073 otu1 u1u3 otot 8013 0709 otu1 u1u9 otot 294 u803 otu2 252 otot u110 u2u6 07 otot 63 otu75212 08 6 otu1 otu1 otu416 239 otu6 u730 otu2 u584 otot 528 u9u8 otot 12 273 otu1 otu649 0597 otu1 09 7 otu2 53 0 otu1 u1 8638 ot u1u9 otot u841 911 ot u306 otu2 u26 574 ot otu7 otu10478 73 ototu4 u9 22 ot u4 900 ot u325 otu2 35 37 ototu6 u8 05 otu102 10 ot u1 91184 ot u1 53 5 ot u1 34 ot u2u15 690 otot 487 2 otu u1 98 4 ottuu12352217 o t u 92 7 o ottu 98 1 o u1 4815 ototuu164913 6 ottuu11216930 o t u1 8 32 o t u 1 5 81 o t u 1 0 99 o t u 1 9 11 o t u1 5 9 3 o t tu 4 05 o o tu 7 41 o tuu7 554 o t u555 8 o t u 83 5 o t u 22 2 o t 1 20 4 o u1 4 3 ottuu22104094 o t u 11 5 9 o t u 13 3 0 o t u u8 1 9 o t t u1 3 5 o o t u507 9 o t 1 3655 o uu 0 4 ototu2u345340 ototu1u8216245 ototu11967403 oottutuu5516626 oot u 1 0 1 ottu1u803437 o otu1124409 ototutuu7 o ot 9 4 26 9 6 u121218 3 ototuu227160 2 ototuu24821 323 oottuu21407061 oottuu21635705 oottuu115032 oottuu1480505 oottuu4181565 oottuu11914294 oottuu222779 0 ottuu92096 4 oot u 97 8 ottuu135832 o t u 39 4 o t u 79 o t u 91 4 o t u 94 4 o t u 54 1 9 o t u 13 1 o t u 10 7 otu 82 o t u 5 6 81 o t u2 6 18 o t u 1 9 54 o t u1 1 3 2 o t u1 8 0 o t u1 3 8 o t u 7 7 67 o t u7 3 8 o t u1 5 o t u7 3 5 o t u4 757 o t u1 41 o t u4 6 o t u6 256 o t u2 02 o t 1 82 o tuu1 869 o t u1 113 o t 1815 0 o u 74 0 ot u1 03 7 ot u1 ot u1 17 7 ot u2 82 ot u1 07 ot u3 60 3 ot u8 79 9 ot u1 04 ot u1 78 ot u6 36 9 ot u5 49 ot u1 66 ot u3 15 ot u5 85 5 ot u3 82 ot u1 7 3 ot u9 65 ot u1 92 ot u2 64 7 ot u8 20 7 ot u2 22 ot u1 43 8 ot u1 68 7 ot u1 11 6 ot u2 80 5 ot u1 13 ot u2 18 3 ot u7 59 ot u1 53 ot u7 1 9 ot u3 14 9 ot u1 80 ot u1 33 ot u4 99 ot u5 4 ot u2 814 ot u1 855 ot u1 695 ot u1 034 ot u1 69 ot u8 81 ot u3 990 ot u1 515 ot u1 24 ot u4 ot u276 ot u941 0 ot u220 2 ot u217 ot u297 1 ot u209 ot u576 3 ot u188 ot u490 5 ot 232 otuu395 4 ot 208 8 otu 155 4 otu 116 9 otu 191 otu 987 otu 401 3 otu 131 otu 219 9 otu 131 otu 479 otu 1686 otu 2040 otu 573 otu 413 otu 1923 otu 2009 otu 2095 otu 06 otu4 01 otu8 536 otu1 341 otu1 05 otu3 94 otu2 519 otu1 60 otu9 20 otu21 80 otu12 73 otu19 otu19 6 otu193 otu859 otu329 otu285 3 otu108 2 otu234 otu2336 otu1196 otu2307 otu1131 otu527 otu1810 otu1896 otu949 otu1036 otu2267 otu1058 otu0 otu727 otu1025 otu1419 otu1456 otu1889 otu2234 otu2176 otu152 otu2199 otu822 otu250 otu1299 otu1787 otu1702 otu1873 otu281 otu805 otu1059 otu316 otu2 otu129 otu217 8 otu180 9 otu409 2 otu18 otu83 08 otu52 5 otu19 0 otu30 52 otu20 6 otu51 87 otu9370 otu84 otu504 otu455 otu514 otu1409 otu1706 otu1328 otu190 otu552 9 otu533 otu229 otu139 6 otu647 2 otu178 otu136 3 otu107 0 otu134 2 otu726 otu145 otu193 5 otu167 7 otu749 otu693 7 otu230 otu62 5 otu4 otu1 394 otu7 65 otu1 54 2 otu2 94 otu2 18 8 otu5 31 1 otu5 45 4 otu4 46 otu2 52 otu7 12 otu1 45 otu8 15 otu1 90 otu8 76 otu1 66 4 otu6 42 otu4 91 8 otu6 63 otu1 69 otu8 06 otu1 61 5 otu7 29 otu1 64 7 otu6 50 otu1 88 8 otu1 29 otu4 22 5 otu1 77 3 otu7 40 ot ot u1 80 ot u7 30 ot u1 12 7 ot u1 38 ot u1 33 5 ot u1 83 0 ot u2 12 9 ot u6 05 4 ot u4 44 7 ot u8 43 ot u1 78 ot u2 47 ot u1 27 4 ot u5 85 1 ot u6 26 7 ot u1 11 ot u8 65 ot u2 83 0 ot u1 10 ot u1 45 0 ot u1 o u2 35 ottu 03 3 ot 8127 o u2 8 3 o tuu8 16 o t 2184 0 o t u5 28 o tuu5 17 o tu 1 64 o t 2 07 o t u1 26 7 o t u4 13 4 ottuu15123 3 o 9 o t u1 3 o t u1 1 57 o t u 7 1 99 o t u2 6 26 o t u3 6 7 o t u1 6 5 o t u9 1 7 o t u1 6 1 2 o t u1 5 2 otu 85 o t u 12 8 1 ottuu11397048 oot u 22 2 5 ottuu21138363 oot u 16 2 1 ottuu21585557 o ottuu34119 6 oottuu121425 oottuu11075904 oottuu2813063 oottuu7893331 oottuu516591 oottuu7618854 oottuu212398 ototuu215 1128 ootuu2130235 8 ototutu317 07434 u1166278 6 4 1 47 2 111 7 112 Fig. 2. 113 Bacteria * Active * A * 114 Fungi * C Dormant B Active D Dormant 115 116 8 117 Fig. 3. Bacteria Fungi A B C D 118 9
