<p>Eubacteria </p><p><em>Fabia U. Battistuzzi</em><sup style="top: -0.2667em;">a,b,</sup><em>* and S. Blair Hedges</em><sup style="top: -0.2667em;">a </sup></p><p>Department of Biology, 208 Mueller Laboratory, The Pennsylvania State University, University Park, PA 16802-5301, USA; <sup style="top: -0.2267em;">b</sup>Current address: Center for Evolutionary Functional Genomics, The Biodesign Institute, Arizona State University, Tempe, AZ 85287-5301, USA *To whom correspondence should be addressed (Fabia.Battistuzzi@ asu.edu) </p><p>shown increasing support for lower-level phylogenetic clusters (e.g., classes and below), they have also shown the susceptibility of eubacterial phylogeny to biases such as horizontal gene transfer (HGT) (20, 21). <br>In recent years, three major approaches have been used for studying prokaryote phylogeny with data from complete genomes: (i) combining gene sequences in a single analysis of multiple genes (e.g.,&nbsp;7, 9, 10), (ii) combining trees from individual gene analyses into a single “supertree” (e.g., 22, 23), and (iii) using the presence or absence </p><p>Abstract </p><p>The ~9400 recognized species of prokaryotes in the&nbsp;of genes (“gene content”) as the raw data to investigate </p><p>Superkingdom Euba cteria&nbsp;a re pla ced in 25 phyla . Their relationships have been difficult to establish, although some ma jor groups a re emerging from genome a na lyses. A molecular timetree, estimated here, indicates that most (85%) of the phyla&nbsp;a nd cla sses a rose in the Archea n Eon (4000−2500 million years ago, Ma) whereas most (95%) of </p><p>relationships (e.g., 17, 18). While the results of these different approaches have not agreed on many details of relationships, there have been some points of agreement, such as support for the monophyly of all major classes and some phyla (e.g., Proteobacteria and Firmicu tes). 7ese Andings, although criticized by some (e.g., 24, 25), </p><p>the families arose in the Proterozoic Eon (2500−542 Ma). It&nbsp;suggest the presence of a detectable evolutionary sigalso points to an early origin of phototrophy (3400–3200 Ma) followed by colonization of land (3041–2755 Ma), origin of oxygenic photosynthesis (2920–2592 Ma) and of aerobic methanotrophy (2630–2371 Ma). </p><p>nal for prokaryotes when using carefully selected genes (e.g., vertically inherited) and appropriate methodologies (e.g., genome-scale data sets). </p><p>7e Su perkingdom Eu bacteria (also called “Bacteria,” Fig. 1) is subdivided into 25 recognized phyla1(), although other classiAcations have been proposed (2) and putative lineages await taxonomic assignment (e.g., 3, 4). In contrast to most eukaryotic groups, eubacterial phylogeny has been di1cult to resolve at the highest level (i.e., phyla and class relationships) despite the availability of many complete genome sequences. 7e small subu&nbsp;nit (SSU) ribosomal RNA (rRNA) gene has been widely used to study prokaryote phylogeny and Arst revealed the distinction of eubacteria from archaebacteria (Archaea) (5, 6). However, becau se of its limited length and the large genetic distances among prokaryote species, it is also subject to biases such as long-branch attraction, base composition diBerences, and a generally poor resolving power. 7ese issues mostly have been addressed in more recent years by using genome-scale data sets (e.g., multiple genes, gene content, and insertion–deletion events) and increased taxonomic sampling (7–19, 88). While these studies have </p><p>Fig. 1&nbsp;<em>Spirillum</em>, a betaproteobacterium (upper left); <em>Beggiatoa</em>, a gammaproteobacterium (upper right); an unidentified cyanobacterium (lower left); and an unidentified spirochete (lower right). Credits: D. J. Patterson, L. Amaral-Zettler, and V. Edgcomb (upper left and right); D. J. Patterson (lower left); and L. Amaral-Zettler, L. Olendzenski, and D. J. Patterson (lower <a href="/goto?url=http://microscope.mbl" target="_blank">right). Images provided by micro*scope (http://microscope.mbl. </a>edu) under creative commons license. </p><p>F. U. Battistuzzi and S. B. Hedges. Eubacteria. Pp. 106–115 inꢀe Timetree of Life, S. B. Hedges and S. Kumar, Eds. (Oxford University Press, New York, 2009). </p><p>Eubacteria </p><p>107 </p><p>Staphylococcaceae Bacillaceae <br>50 54 <br>61 <br>Listeriaceae <br>30 </p><p>29 <br>Lactobacillaceae Streptococcaceae Acholeplasmataceae Mycoplasmataceae-1 Mycoplasmataceae-2 Entomoplasmataceae </p><p><em>Symbiobacterium Carboxydothermus </em></p><p>Peptococcaceae <br>21 <br>46 <br>85 <br>15 </p><p>24 <br>33 <br>44 <br>20 </p><ul style="display: flex;"><li style="flex:1">8</li><li style="flex:1">Thermoanaerobacteriaceae-1 </li></ul><p>Clostridiaceae <br>36 <br>Thermoanaerobacteriaceae-2 </p><p><em>Dehalococcoides </em></p><p>Gloeobacteraceae Synechococcaceae-1 Synechococcaceae-2 Synechococcaceae-3 Nostocaceae <br>11 <br>60 </p><p>66 <br>6<br>75 </p><p>76 <br>80 <br>Merismopediaceae </p><p>Deinococcaceae Propionibacteriaceae Frankiaceae <br>53 </p><ul style="display: flex;"><li style="flex:1">63 </li><li style="flex:1">13 </li><li style="flex:1">Corynebacteriaceae </li></ul><p>Mycobacteriaceae Nocardiaceae <br>82 <br>88 <br>59 </p><p>43 <br>Streptomycetaceae </p><p>Nocardiopsaceae Bifidobacteriaceae Cellulomonadaceae Microbacteriaceae <br>69 <br>48 <br>71 </p><p><em>(continued on next page) </em></p><p></p><ul style="display: flex;"><li style="flex:1">Ea Pa Ma&nbsp;Na </li><li style="flex:1">Pp </li><li style="flex:1">Mp </li><li style="flex:1">Np Pz </li></ul><p>PH <br>HD <br>ARCHEAN </p><p>3000 </p><p>Fig. 2&nbsp;<em>Continues </em></p><p>PROTEROZOIC </p><ul style="display: flex;"><li style="flex:1">2000 1000 </li><li style="flex:1">4000 </li><li style="flex:1">0 Million&nbsp;years ago </li></ul><p></p><p>108 </p><p>THE TIMETREE OF LIFE </p><p><em>(continued from previous page) </em></p><p>Francisellaceae Shewanellaceae Vibrionaceae <br>73 <br>77 <br>81 <br>Enterobacteriaceae Pasteurellaceae Idiomarinaceae Colwelliaceae <br>42 <br>70 </p><p>74 <br>79 <br>47 <br>Pseudoalteromonadaceae </p><p>Moraxellaceae <br>37 <br>57 <br>Pseudomonadaceae </p><p>Hahellaceae <br>67 <br>32 <br>Coxiellaceae </p><p>51 <br>Legionellaceae </p><p>Chromatiaceae <br>38 </p><p>34 <br>Methylococcaceae Piscirickettsiaceae Xanthomonadaceae Neisseriaceae <br>4<br>26 <br>39 </p><p>Rhodocyclaceae Comamonadaceae Burkholderiaceae Alcaligenaceae <br>55 <br>68 </p><p>72 <br>78 <br>58 </p><p>Nitrosomonadaceae Hydrogenophilaceae <br>64 </p><p><em>(continued on next page) </em></p><p></p><ul style="display: flex;"><li style="flex:1">Ea Pa Ma&nbsp;Na </li><li style="flex:1">Pp </li></ul><p>PROTEROZOIC </p><p>2000 1000 </p><ul style="display: flex;"><li style="flex:1">Mp </li><li style="flex:1">Np Pz </li></ul><p>HD <br>ARCHEAN </p><p>3000 </p><p>Fig. 2&nbsp;<em>Continues </em></p><p>PH </p><ul style="display: flex;"><li style="flex:1">4000 </li><li style="flex:1">0 Million&nbsp;years ago </li></ul><p></p><p>Phylogenetic analyses that have focused on subgroups&nbsp;ribosomal proteins, DNA-directed RNA polymerase of prokaryotes (e.g., classes) also have su pported, consistently, particular groupings. For example, cyanobacteria and low GC gram positives (Firmicutes) have been subunits (30), genome trees (31), and gene content analysis (18). Other taxa, such as AquiAcae and 7ermotogae, have been more di1cult to place, appearing at the base united based on maximum likelihood (ML) mapping of&nbsp;of the tree in some analyses (4, 7, 9, 32) and in a higher, 21 orthologous genes (26); Fusobacteria is the most closely&nbsp;nested position in other analyses (10, 33, 34). related group to Firmicutes based on combined analyses of SSU and large su bu nit (LSU) rRNA sequ ences and <br>Generally concordant relationships were found between past studies and a recent ML and Bayesian anaribosomal proteins (27); Bacteroidetes, Chlorobi, and&nbsp;lysis of multiple core gene sequences (88). In that study, Fibrobacteres form a group based on insertion–deletion&nbsp;a complete matrix of 25 vertically inherited orthologous analysis (28, 29); and Planctomycetacia, Chlamydiae,&nbsp;genes shared by 197 fully sequenced eubacteria and 21 </p><ul style="display: flex;"><li style="flex:1">and Spirochaetes form a group based on concatenated </li><li style="flex:1">fully sequenced species of archaebacteria was built. </li></ul><p></p><p>Eubacteria </p><p>109 </p><p><em>(continued from previous page) </em></p><p>Erythrobacteraceae Rhodobacteraceae Caulobacteraceae Bradyrhizobiaceae Rhizobiaceae <br>17 <br>41 </p><p>45 </p><p>56 <br>65 <br>35 <br>86 <br>84 <br>87 <br>Phyllobacteriaceae Bartonellaceae <br>14 <br>Brucellaceae </p><p>25 <br>Rhodospirillaceae </p><p>Acetobacteraceae SAR11 <br>49 <br>3<br>27 <br>40 <br>Anaplasmataceae Rickettsiaceae Solibacteraceae Desulfovibrionaceae Myxococcaceae Bdellovibrionaceae Pelobacteraceae Geobacteraceae Campylobacteraceae Helicobacteraceae Chlamydiaceae Chlorobiaceae <br>9<br>16 </p><p>18 <br>28 </p><p>22 <br>52 </p><p>5<br>62 <br>2</p><p>12 <br>23 <br>Crenotrichaceae </p><p>Bacteroidaceae Porphyromonadaceae Planctomycetaceae Spirochaetaceae Leptospiraceae <br>31 <br>7<br>83 </p><p>1</p><p>10 <br>19 </p><p>Fusobacteriaceae Aquificaceae Thermotogaceae </p><p>Ea Pa Ma&nbsp;Na ARCHEAN </p><ul style="display: flex;"><li style="flex:1">Pp </li><li style="flex:1">Mp </li><li style="flex:1">Np Pz </li></ul><p>HD <br>PROTEROZOIC </p><p>2000 1000 <br>PH </p><ul style="display: flex;"><li style="flex:1">4000 </li><li style="flex:1">3000 </li><li style="flex:1">0 Million&nbsp;years ago </li></ul><p></p><p>Fig. 2&nbsp;A timetree of eubacteria. Divergence times are shown in Table 1. Codes for paraphyletic and/or polyphyletic groups are as follows: Mycoplasmataceae-1 </p><p>(<em>Mycoplasma genitalium</em>), Mycoplasmataceae-2 (<em>Mycoplasma capricolum</em>), Synechococcaceae-1 (<em>Synechocococcus </em></p><p>JA-2-3Ba), Synechococcaceae-2 (<em>Thermosynechococcus </em></p><p><em>elongatus</em>), Synechococcaceae-3 (<em>Synechococcus elongatus</em>), </p><p>Thermoanaerobacteriaceae-1 (Moorella thermoacetica), Thermoanaerobacteriaceae-2 (Thermoanaerobacter tengcongensis). <em>Abbreviations</em>: Ea (Eoarchean), HD (Hadean), Ma (Mesoarchean), Mp (Mesoproterozoic), Na (Neoarchean), Np (Neoproterozoic), Pa (Paleoarchean), PH (Phanerozoic), Pp (Paleoproterozoic), and Pz (Paleozoic). </p><p>110 </p><p>THE TIMETREE OF LIFE </p><p>Table 1.&nbsp;Divergence times (Ma) and their confidence/credibility intervals (CI) among eubacteria. </p><p></p><ul style="display: flex;"><li style="flex:1">Timetree Estimates&nbsp;Timetree </li><li style="flex:1">Estimates </li></ul><p></p><ul style="display: flex;"><li style="flex:1">Node </li><li style="flex:1">Time </li><li style="flex:1">Ref. (7) </li><li style="flex:1">This study </li></ul><p>CI </p><ul style="display: flex;"><li style="flex:1">Node </li><li style="flex:1">Time </li><li style="flex:1">Ref. (7) </li><li style="flex:1">This study </li></ul><p></p><ul style="display: flex;"><li style="flex:1">CI </li><li style="flex:1">Time </li><li style="flex:1">CI </li><li style="flex:1">Time </li><li style="flex:1">Time </li><li style="flex:1">CI </li><li style="flex:1">Time </li></ul><p></p><p></p><ul style="display: flex;"><li style="flex:1">1</li><li style="flex:1">4189 </li></ul><p>4179 3306 3134 2979 2908 2897 2874 2849 2762 2761 2739 2739 2687 2607 2579 2504 2421 2339 2281 2233 2173 2099 2047 2042 1993 1919 1899 1860 1837 1834 1806 1775 1753 1753 1747 1673 1653 1621 1620 </p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li></ul><p>–<br>4189 4179 3306 3134 2979 2908 2897 2874 2849 2762 2761 2739 2739 2687 2607 2579 2504 2421 2339 2281 2233 2173 2099 2047 2042 1993 1919 1899 1860 1837 1834 1806 1775 1753 1753 1747 1673 1653 1621 1620 </p><ul style="display: flex;"><li style="flex:1">4200–4159 </li><li style="flex:1">45 </li></ul><p>46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 <br>1498 1482 1481 1436 1432 1429 1420 1413 1402 1392 1386 1326 1306 1224 1189 1180 1121 1104 1069 1055 1042 1037 1030 1028 1027 950 </p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li></ul><p>–<br>1498 1482 1481 1436 1432 1429 1420 1413 1402 1392 1386 1326 1306 1224 1189 1180 1121 1104 1069 1055 1042 1037 1030 1028 1027 950 <br>1644–1351 1656–1313 1586–1372 1600–1270 1580–1283 1597–1269 1539–1297 1573–1257 1557–1241 1573–1223 1511–1262 1471–1183 1420–1190 1349–1101 1332–1040 1334–1035 1276–977 1271–943 1209–928 1184–932 1180–912 1186–902 1145–917 1150–911 1167–887 1051–849 1084–794 993–759 </p><ul style="display: flex;"><li style="flex:1">2</li><li style="flex:1">–</li><li style="flex:1">4197–4141 </li></ul><p>3447–3165 3265–2987 3116–2835 3041–2755 3040–2747 3012–2716 2983–2706 2926–2595 2920–2592 2894–2575 2897–2570 2815–2549 2753–2448 2715–2428 2630–2371 2563–2267 2517–2154 2439–2115 2401–2067 2321–2018 2261–1932 2215–1873 2187–1887 2099–1894 2083–1749 2078–1719 2040–1680 2011–1673 2005–1665 1889–1731 1948–1602 1821–1690 1894–1605 1931–1572 1765–1581 1688–1640 1722–1520 1792–1449 <br>–</p><ul style="display: flex;"><li style="flex:1">3</li><li style="flex:1">3186 </li><li style="flex:1">3634–2801 </li><li style="flex:1">1387 </li><li style="flex:1">1763–1060 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">4</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">5</li><li style="flex:1">3096 </li><li style="flex:1">3539–2723 </li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">6</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">1561 </li><li style="flex:1">2012–1166 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">7</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">8</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">9</li><li style="flex:1">2800 </li><li style="flex:1">3223–2452 </li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p>10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 </p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">1470 </li><li style="flex:1">1873–1114 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">1537 </li><li style="flex:1">1942–1151 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">2688 </li><li style="flex:1">3108–2360 </li><li style="flex:1">1380 </li><li style="flex:1">1798–1038 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">2508 </li><li style="flex:1">2928–2154 </li><li style="flex:1">1282 </li><li style="flex:1">1690–916 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">1244 </li><li style="flex:1">1694–856 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">2305 </li><li style="flex:1">2729–1944 </li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">2030 </li><li style="flex:1">2449–1656 </li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">1945 </li><li style="flex:1">2368–1573 </li><li style="flex:1">–</li><li style="flex:1">–</li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">937 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">937 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">872 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">872 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">871 </li><li style="flex:1">–</li></ul><p>–</p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">871 </li><li style="flex:1">972–772 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">1816 </li><li style="flex:1">2261–1415 </li><li style="flex:1">812 </li><li style="flex:1">–</li><li style="flex:1">812 </li><li style="flex:1">910–711 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">793 </li><li style="flex:1">1039 </li></ul><p>–</p><ul style="display: flex;"><li style="flex:1">1408–702 </li><li style="flex:1">793 </li><li style="flex:1">923–678 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">1751 </li><li style="flex:1">2163–1390 </li><li style="flex:1">751 </li><li style="flex:1">–</li><li style="flex:1">751 </li><li style="flex:1">880–639 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">854–653 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">751 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">751 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">744 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">744 </li><li style="flex:1">859–640 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">668 </li><li style="flex:1">–</li><li style="flex:1">–</li><li style="flex:1">668 </li><li style="flex:1">768–573 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">2132 </li><li style="flex:1">2552–1760 </li><li style="flex:1">662 </li><li style="flex:1">756 </li></ul><p>–</p><ul style="display: flex;"><li style="flex:1">1070–484 </li><li style="flex:1">662 </li><li style="flex:1">783–553 </li></ul><p>––––<br>––––</p><ul style="display: flex;"><li style="flex:1">634 </li><li style="flex:1">–</li><li style="flex:1">634 </li><li style="flex:1">739–538 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">621 </li><li style="flex:1">928 </li></ul><p>–</p><ul style="display: flex;"><li style="flex:1">1274–644 </li><li style="flex:1">621 </li><li style="flex:1">734–516 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">616 </li><li style="flex:1">–</li></ul><p>–</p><ul style="display: flex;"><li style="flex:1">616 </li><li style="flex:1">734–506 </li></ul><p></p><ul style="display: flex;"><li style="flex:1">594 </li><li style="flex:1">–</li><li style="flex:1">594 </li><li style="flex:1">699–499 </li></ul><p></p><p>Eubacteria </p><p>111 </p><p>Table 1.&nbsp;<em>Continued </em></p><p></p><ul style="display: flex;"><li style="flex:1">Timetree </li><li style="flex:1">Estimates </li><li style="flex:1">Timetree </li><li style="flex:1">Estimates </li></ul><p></p><ul style="display: flex;"><li style="flex:1">Node </li><li style="flex:1">Time </li><li style="flex:1">Ref. (7) </li><li style="flex:1">This study </li></ul><p>CI </p><ul style="display: flex;"><li style="flex:1">Node </li><li style="flex:1">Time </li><li style="flex:1">Ref. (7) </li><li style="flex:1">This study </li></ul><p></p><ul style="display: flex;"><li style="flex:1">Time CI </li><li style="flex:1">Time </li><li style="flex:1">CI </li><li style="flex:1">Time </li><li style="flex:1">Time </li><li style="flex:1">CI </li></ul><p></p><p>41 42 43 44 <br>1613 1612 1579 1554 <br>––––<br>––––<br>1613 1612 1579 1554 </p><ul style="display: flex;"><li style="flex:1">1761–1465 </li><li style="flex:1">85 </li></ul><p>86 87 88 <br>523 509 432 380 <br>––––<br>––––<br>523 509 432 380 <br>638–425 609–421 520–351 469–304 <br>1712–1506 1740–1411 1729–1378 </p><p>Note: Nodes times in the timetree are from this study. </p><p>Single-gene phylogenies were screened for orthology&nbsp;supported in the Bayesian phylogeny as well. Multiple and vertical inheritance under the assumption that phy-&nbsp;evidence from other analyses (2, 11, 23, 28) supports logenies showing mixing of the two superkingdoms or signiAcantly supported deep nesting of one class within <br>Bacterobi, while Spiroplancti is present in some but not all previous studies. However, currently no clear physioanother were aBected by HGT and deleted from the&nbsp;logical or metabolic adaptations can be considered as data set. Site homology of the multiple sequence alignment was established with GBlocks (35) and nonconunifying characteristics of these clusters. <br>Molecular clock methods have been used infrequently served sites were deleted. ML (36) and Bayesian (37) with&nbsp;eubacteria, probably because of di1culties in estabmethods were then applied to the Anal alignment (6884&nbsp;lishing a robust phylogeny and in selecting reliable calisites) to estimate phylogenetic relationships. A ML phyl-&nbsp;bration points. Calibrations have included the fossil </p><ul style="display: flex;"><li style="flex:1">ogeny was also constru cted from an alternative align- </li><li style="flex:1">record of prokaryotes and eukaryotes and biomarkers </li></ul><p>ment with only slow-evolving positions (16,344 sites)&nbsp;in the geologic record. A problem with most eukaryote and showed a similar phylogeny (except for the pos-&nbsp;fossils, otherwise useful for calibration, is that they are ition of Solibacteres). A topological feature common to&nbsp;from the Phanerozoic eon (543–0 Ma), mu ch you nger </p><ul style="display: flex;"><li style="flex:1">all of these analyses is a major dichotomy in eubacteria. </li><li style="flex:1">than many key nodes of interest to time. 7is exac- </li></ul><p>7is is formed by the Terrabacteria (Actinobacteria, erbates&nbsp;the problems with diBerent rates of evolu tion ChloroPexi, Cyanobacteria, Deinococcus-ꢀermus, and&nbsp;among the three superkingdoms (38), an issue dimin- </p><ul style="display: flex;"><li style="flex:1">Firmicu tes)&nbsp;and the Hydrobacteria (Acidobacteria, </li><li style="flex:1">ished by using calibrations within each superkingdom. </li></ul><p>Bacteroidetes, Chlamydiae, Chlorobi, Planctomycetacia,&nbsp;Although the prokaryote fossil record extends reliably Proteobacteria, and Spirochaetes) to the exclusion of the&nbsp;back to ~2000 Ma (39), the taxonomic resolution is not hyperthermophiles and, possibly, Fusobacteria (88). 7is&nbsp;su1cient in most cases to be broadly useful for calibradeAnition of Terrabacteria is more inclusive than the ini-&nbsp;tion. Better resolution for a few groups is obtained from tial deAnition provided in 2004, which did not include&nbsp;the geologic record with biomarkers produced speciAcChloroPexi and Firmicutes (7). <br>In addition, three possible high-level clusters within <br>Hydrobacteria are highly supported by the ML phylally and exclusively by one lineage (40). Prokaryotes that are parasitic on eukaryotes provide other opportunities for calibration (41, 42), but they are almost entirely in the ogeny. Bacteroidetes, Chlorobi, Chlamydiae, Plancto-&nbsp;Phanerozoic, again much younger than many deep nodes mycetes, and Spirochaetes are associated in a single&nbsp;of interest. One additional calibration that has been used </p><ul style="display: flex;"><li style="flex:1">clu ster, with high bootstrap su pport (82%), for which </li><li style="flex:1">is the maximum time for life on Earth (7), which can be </li></ul><p>we propose the name Spirochlamydiae. Within this&nbsp;set either to the origin of Earth at 4600 Ma as an absoinfrakingdom, two other superphyla are strongly sup-&nbsp;lute maximum or perhaps more realistically to the last ported: (i) Planctomycetes and Spirochaetes (93%) and&nbsp;ocean-vaporizing event, ~4200 Ga (43). </p><ul style="display: flex;"><li style="flex:1">(ii) Bacteroidetes and Chlorobi (100%). For these we </li><li style="flex:1">Data sets using diBerent types of molecular informa- </li></ul><p>propose the names Spiroplancti and Bacterobi, respect-&nbsp;tion (i.e., rRNA, enzymes, core genes) have been used to ively. 7ese last two clusters are present and signiAcantly&nbsp;estimate divergence times. A divergence time analysis of </p>