phylogenetic analyses with systematic taxon sampling show ...10.1038... · 20 and compositional...

Articleshttps://doi.org/10.1038/s41559-020-1239-x

Phylogenetic analyses with systematic taxon sampling show that mitochondria branch within AlphaproteobacteriaLu Fan 1,2,3,7 ✉, Dingfeng Wu4,7, Vadim Goremykin5,7, Jing Xiao4, Yanbing Xu4, Sriram Garg 6, Chuanlun Zhang1,3, William F. Martin 6 ✉ and Ruixin Zhu 4 ✉

1Shenzhen Key Laboratory of Marine Archaea Geo-Omics, Department of Ocean Science and Engineering, Southern University of Science and Technology (SUSTech), Shenzhen, China. 2Academy for Advanced Interdisciplinary Studies, Southern University of Science and Technology (SUSTech), Shenzhen, China. 3Southern Marine Science and Engineering Guangdong Laboratory, Guangzhou, China. 4Department of Bioinformatics, Putuo People’s Hospital, Tongji University, Shanghai, China. 5Research and Innovation Centre, Fondazione Edmund Mach, San Michele all’Adige, Italy. 6Institute of Molecular Evolution, Heinrich Heine University, Düsseldorf, Germany. 7These authors contributed equally: Lu Fan, Dingfeng Wu, Vadim Goremykin. ✉e-mail: [email protected]; [email protected]; [email protected]

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Supplementary Information 1

This file contains Supplementary Notes 1–9, Supplementary Methods, Supplementary Figures 1–60, and 2 Supplementary References. 3

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Table of Contents 5

Supplementary Notes………02 6

Supplementary Methods…...07 7

Supplementary Figures…….09 8

Supplementary References...69 9

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Supplementary Notes 11

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Supplementary Note 1 13

Difficulties in resolving the phylogenetic relationship with extant alphaproteobacterial lineages (see a 14 detailed review in reference 5). (1) Considerable phylogenetic divergence and metabolic variety within 15

Alphaproteobacteria1; (2) faint historical signals left behind the very ancient event of mitochondria origin2; 16 (3) limited number of marker genes shared between mitochondria and Alphaproteobacteria due to extensive 17

gene loss in the prior3; (4) taxonomic bias in datasets towards clinically or agriculturally important 18 alphaproteobacterial members4; and (5) strong phylogenetic artefacts such as long-branch attraction (LBA) 19

and compositional heterogeneity associating mitochondria with fast-evolving alphaproteobacterial lineages. 20

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Heterogeneity mitigation methods. As datasets in studies on phylogeny between mitochondria and 23

Alphaproteobacteria heavily suffer from compositional heterogeneity and LBA, various approaches to 24 mitigate non-historical signals have been adopted but the drawbacks of these methods are rarely examined. 25 Among them, protein recoding cause signal loss and artificial mutation saturation6. Nucleus-encoded 26

mitochondrial genes have to be adapted to new rules of expression and regulation in the nucleus system and 27 therefore may actually have undergone intensive site substation compared to mitochondrion-encoded genes. 28

Thus, the reliability of using nucleus-encoded mitochondrial genes in phylogenetic analysis of mitochondria 29 need further justification7,8. The idea of excluding potentially model-violating sites to improve phylogenetic 30

prediction was introduced over two decades ago9,10 but has been opposed by some researchers (see review 31 by Shepherd and Klaere11). The concern is that in spite of non-historical signals, these sites may contain 32

useful information. Nonetheless, various versions of site exclusion have been applied in phylogenetic studies 33 of mitochondria and Alphaproteobacteria either based on evolving rate12,13 or amino acid composition14,15,16. 34

However, conflicting results were reported by using different site-exclusion metrics17. 35

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Explanations and verifications to the topological shift of trees observed in Martijn et al. (2018). In the 38 study of Martijn et al.16, it was observed that when certain amount of sites in the alignment matrix were 39

excluded by using either a Stuart-score based stationary trimming method or a !2-score based method, the 40

tree topology shifted from supporting Rickettsiales-sister to Alphaproteobacteria-sister for mitochondria. At 41 least two mechanisms may explain this observation: (1) Mitochondria originated independently from extant 42

Alphaproteobacteria. The affinity between mitochondria and Rickettsiales in phylogenetic trees is the result 43 of convergent evolution in amino acid composition instead of common evolutionary history. Site-exclusion 44

approaches filter out this non-historical signal by removing the most heterogenous sites in datasets and 45 restore the true phylogenetic position of mitochondria; and (2) Historical signals between Mitochondria and 46

some alphaproteobacterial groups exist but are weak as the result of divergent evolution for billions of years. 47

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Site-exclusion approaches employed by Martijn et al. removed most of the sites containing these signals and 48 broke the true evolutionary connection between mitochondria and Alphaproteobacteria. The long branch of 49 mitochondria in the phylogenetic tree is then attracted by the long branch leading towards the outgroup taxa 50

containing other proteobacteria resulting an Alphaproteobacteria-sister topology. However, since there is a 51 lack of known close relatives to the entire Alphaproteobacteria clade, it is impossible to validate the LBA 52

effect in this tree by replacing the distant outgroup with a much closer alternative (but see the placement of 53 mitochondria in the Alpha IIb with Alpha IIa as a short-branched outgroup in this study). 54

Martijn et al. testified the potent LBA effect of outgroup taxa by conducting three analyses16. As admitted 55 by them, the outgroup removal analysis did not proof or disproof the hypothesis of LBA by the outgroup. 56

Secondly, the parametric simulation approach they conducted generated ten trees in which eight recovered 57 a Rickettsiales-sister topology with weak node supports (below 80%) (Supplementary Figure 20 in their 58

publication). As suggested by the IQ-TREE manuscript17, Ultra-Fast bootstrapping values below 95% are 59 unreliable. Therefore, the result they obtained may actually suggest that a trustable Rickettsiales-sister 60

topology was unable to be recovered and LBA effect of the outgroup might have a function on it. Lastly, the 61 authors declared that random sequence replacements showed that the long branch of outgroup did not attract 62 any of the random sequences. However, this is completely untrue! In the ten trees they provided as 63

Supplementary Data, there are 8, 0, 3, 8, 5, 0, 0, 6, 0, and 3 of the 10 random sequences attracted by the 64 outgroup, respectively. Therefore, the authors failed to exclude the possible artificial attraction of outgroup 65

to mitochondria after site exclusion. 66

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Notable inconsistence in data reproducibility in the study of Martijn et al.16. In addition to the 69

misinterpretation of results by Martijn et al. we mentioned in Supplementary Note 3, we here report two 70 cases of data irreproducibility in Martijn et al. (2018). First, the Bayesian consensus tree we obtained of the 71

untreated ‘24-alphamitoCOGs’ dataset (Supplementary Figure 1) is fundamentally different from the ones 72 show in Supplementary Figure 9 in Martijn et al. (2018), albeit we used the same dataset and tree 73

construction parameters as they did. Specifically, in our tree, fast-evolving taxonomic groups do not form 74 monophyletic groups. Instead, some taxa such as Pelagibacter were placed with slow-evolving 75 alphaproteobacteria. Mitochondria and Rickettsiales formed a monophyletic clade, which is in adjacent by 76

other alphaproteobacterial groups. Thus, our result shows notable capacity of the CAT model in dealing with 77 phylogenetic artefacts. 78

Second, the results of posterior predictive tests to the Bayesian inference of the site-excluded dataset based 79 on Stuart’s test (this tree is the cornerstone to support the Alphaproteobacteria-sister conclusion of 80

mitochondria by Martijn et al., Supplementary Table 2) are not comparable to the ones reported by Martijn 81 et al. (Supplementary Figure 11 and Supplementary Table 3 in reference 16). Specifically, they reported a 82

much better model fit according to the maximum squared heterogeneity (Z-score 0.5 – 0.6 in their study, but 83 around 3.09 in this study; P-value 0.25 – 0.27 in their study, but around 0.01 in this study) and mean squared 84

heterogeneity (Z-score -2.5 – -2.3 in their study, but around 2.67 in this study; P-value 0.98 – 0.99 in their 85

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study, but around 0.004 in this study). We are not sure why such inconsistent results were obtained in these 86 two studies since (1) we used the same dataset (the dataset was directly provided by Martijn) and tree 87 reconstruction protocol; and (2) we both obtain the almost same consensus tree topology (Supplementary 88

Figure 2 in this study and Supplementary Figure 11 in their study). The only difference that may explain the 89 above two cases of inconsistency may be that different versions of PhyloBayes MPI were used in the two 90

studies (i.e. version 1.8 here and 1.7a in their study). 91

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Sequence heterogeneity and taxa selection of the ‘Modified18’ dataset. In the ‘Modified18’ dataset, GC-94

rich mitochondrial sequences were selected to remarkably reduce the heterogeneity in FYMINK/GARP ratio 95 between mitochondria and slowly evolving alphaproteobacteria (Supplementary Figure 24). High GC taxa 96

of Rickettsiales taxa were also selected with higher genome G&C content in comparison to those in the ‘24-97 alphamitoCOGs’ dataset. The rationale behind this selection is that the phylogenetic position of Rickettsiales 98

to the backbone taxa of alphaproteobacteria is difficulty to resolve (see Supplementary Note 7). 99 Rickettsiales with low G&C content may artificially attracted by other fast-evolving species in the tree 100 leading to the loss of weak phylogenetic connection to the backbone taxa. 101

It is necessary to notice that the introduced GC-rich mitochondria were all from higher plants. While this 102 may compromise the representation of data, it has been noticed as early as in 1980s by Carl Woese et al. that 103

the mitochondrial sequences of higher plants may have diverged from bacterial sequences to low extent18-20, 104 possibly as a result of low mutation rate in genes maintained by DNA repair mechanisms21. With this in 105

concern, plant mitochondria are as suitable as those of jakobids for phylogenetic analysis with bacterial 106 sequences in term of branch length. Indeed, branch lengths of plant mitochondria in trees based on the 107

‘Modified18’ dataset are comparable to those of single cell eukaryotes based on the ‘Modified24’ dataset as 108 shown in this study (Supplementary Figures 31–36). 109

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The four backbone clades. Group GT (Supplementary Table 3) is equivalent to Geminicoccaceae in 112 Muñoz-Gómez et al. (2019)15. Alpha I comprises core Alphaproteobacterial orders including 113 Kordiimonadales, Sphingomonadales, Rhizobiales, Caulobacterales, Parvularculales and Rhodobacterales. 114

Grouping of these lineages is in consistence with the findings by Muñoz-Gómez et al. and others1,15,22. Alpha 115 II comprises three isolates belonging to Rhodospirillaceae and several marine alphaproteobacterial 116

metagenome-assembled genomes (MAGs). Grouping of these lineages was observed in by Williams et al. 117 and others1,15,22. Alpha III comprises Kiloniellaceae, SAR116, Acetobacteraceae, Azospirillaceae, and some 118

taxa classified to the polyphyletic Rhodospirillaceae. This result is similar to the finding by Muñoz-Gómez 119 et al.15. 120

Notably, separation of these four groups were exactly recovered by Martijn et al. in their untreated ‘24-121 alphamitoCOGs’ dataset (Supplementary Figure 9, 10 in Martijn et al. (2018)), but not in their stationary-122

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trimmed dataset (Fig. 4a and Supplementary Figure 11, 12 in Martijn et al. (2018)), which they claimed to 123 support their ‘mito-out’ result. This again suggests site exclusion may result in abnormal tree topology for 124 even slow-evolving species. 125

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Phylogenetic positions of fast-evolving alphaproteobacterial lineages. Holosporales was previously 128 considered as a subclade of Rickettsiales based on phylogenetic analysis and the factor that members of both 129

groups are obligate endosymbionts23-25. However, other studies suggested the phylogenetic affinity between 130 Holosporales and other Rickettsiales families is the result of artifact16,23,26-28. In the very recent study by 131

Muñoz-Gómez et al. where amino acid bias was corrected by site exclusion, Holosporales was suggested to 132 have a derived position within the Rhodospirillales and possibly close to Azospirllaceae15. Here, 133

Holosporales are in sister-relationship with the entire Alpha III (Fig. 2cd). Our results support that 134 Holosporales are independent of Rickettsiales but may be close to taxa in Alpha III. Recent studies suggested 135

the grouping of Pelagibacterales, alpha proteobacterium HIMB59 and Rickettsiales, as reported by many 136 earlier studies is the result of a compositional bias artefact15,16. Using site-excluded datasets, it was suggested 137 that Pelagibacterales should be placed after the common ancestor of Sphingomonadales (belonging to Alpha 138

Ia here) but before the divergence of Rhodobacterales, Caulobacterales and Rhizobiales (belonging to Alpha 139 Ib here)15. Our result is consistent with this (Fig. 2ef). Moreover, the exact phylogenetic position of alpha 140

proteobacterium HIMB59 could not be resolved by itself (Fig. 2gh). When mitochondria were present, it is 141 placed in the Alpha IIb clade based on the ‘Modified18’ dataset (Fig. 3f). Rickettsiales appearing as sister to 142

all other alphaproteobacteria has been reported in some artefact-attenuated studies15 while conflicting results 143 were recovered in others16 suggesting the current difficulty in resolving its relationship with slow-evolving 144

alphaproteobacteria. We found that Rickettsiales were placed within Alpha II, as the sister of MarineAlpha9 145 Bin5 based on the ‘Modified18’ dataset (Fig. 2i). 146

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Evidences that the tree topology in Fig. 4 is not the results of artefact. First, taxon-exclusion analyses 149 clearly demonstrate the phylogenetic connections of fast-evolving alphaproteobacterial lineages including 150 Rickettsiales and fast-evolving MAGs (FEMAGs) to slow-evolving taxa MarineAlpha9 Bin5 and 151

MarineAlpha11 in the absence of possible influence from non-historical signals (Fig. 2ij). Secondly, 152 mitochondria and these fast-evolving taxa do not form a singlet clade falling apart from backbone clades as 153

a result of LBA – something shown in Supplementary Figure 9, 10 in Martijn et al. (2018). Instead, they 154 were placed with slow-evolving taxa within Alpha IIb (Fig. 3kl and 4). Lastly, taxon-reduction and site-155

exclusion examinations on the datasets ‘Modified24-AlphaII’, ‘Modified24-AlphaII-MoreTaxa’ and 156 ‘Modified18-AlphaII’ as shown in Fig. 1d suggest their tree topology is robust. 157

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Metabolic analysis. Metabolism reconstruction of the bacterial ancestor of mitochondria is essential to 160 syntrophic-based models of eukaryogenesis29-31. Proteome study of alphaproteobacterial sister of 161 mitochondria might provide hints to the metabolic nature of the latest common ancestor of mitochondria and 162

Alphaproteobacteria particularly for those proteins ultimately having been lost in extant mitochondria or 163 nuclei31,32. However, as evidenced by our previous studies33,34, extensive horizontal gene transfer events 164

have been taken place in the 1.8 billion years’ evolutionary history of extant alphaproteobacteria since 165 eukaryogenesis. Consequently, few proteins in extant alphaproteobacteria other than those used for 166

phylogeny here (the 24 marker proteins including riboproteins) were acquired through vertical transfer from 167 the latest common ancestor of mitochondria and Alphaproteobacteria. We therefore think approaches 168

inferring the very ancient event of eukaryogenesis based on proteomes of extant Alphaproteobacteria without 169 robust justification of each protein’s evolutionary pathway is prone to systematic errors. 170

Nevertheless, we here test two hallmarks of metabolic characters in the bacterial ancestor of mitochondria 171 predicted by the ‘hydrogen hypothesis’30 – aerobic and anaerobic oxidation of pyruvate and hydrogen 172

production – by annotating proteomes of taxa in the ‘24-alphamitoCOGs’ dataset. Pyruvate dehydrogenase 173 E1 component alpha subunit (PdhA) involving in aerobic pyruvate oxidation is found in almost all the 174 alphaproteobacteria but only one species in the outgroup (Supplementary Table 7). In contrast, one of the 175

pyruvate ferredoxin oxidoreductases PorA is only detected in Magnetospirillum magneticum, while the other 176 two, OorA and IorA, are found generally in the Alpha II subclade including FEMAG I, some Alpha I and 177

Alpha II taxa and two outgroup taxa. The result suggests ubiquitous aerobic pyruvate oxidation but 178 sporadically distributed anaerobic pyruvate oxidation in extant alphaproteobacteria. NiFe hydrogenase HoxF 179

is only encoded by M. magneticum and two outgroup taxa, probably because most taxa studied here are 180 better adapted to aerobic environments. 181

To investigate the possible evolutionary pathways of these enzymes, phylogenetic trees were reconstructed 182 for PdhA, OorA and IorA (Supplementary Figures 59 and 60). In general, all the trees are in low resolution 183

as shown by nearly half of the nodes with unstable supports (ultra-fast bootstrapping values < 95%) likely 184 caused by short sequence length of each protein. In addition, possible systematic errors such as 185

compositional heterogeneity and LBA may bias the results. Despite, in branches with stable supports, we 186 observe both evidences of local vertical transfer as shown by small monophyletic subclades (e.g. PdhA in 187 Rickettsiales, FEMAG II and Alpha Ia, respectively, Supplementary Figure 59) and lateral transfer as 188

demonstrated by polyphyletic subclades. Overall, it is likely that the three enzymes representing 189 aerobic/anaerobic pyruvate oxidation were present in the common ancestors of all extant alphaproteobacteria 190

and experienced differential loss, duplication and sporadically lateral exchanges with other bacteria. 191 However, since the low resolution of the trees, it is impossible to trace the basal nodes of Alpha II taxa. No 192

evidence of pyruvate oxidation or hydrogen production for the common ancestor of mitochondria and Alpha 193 II taxa can be obtained based on this very limited data. 194

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Supplementary Methods 197

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The site-exclusion method based on the score of Bowker’s test. Compared to Stuart’s test, Bowker’s test 199

of symmetry was reported to more comprehensive and sufficient to assess the compliance of symmetry, 200 reversibility and homogeneity in time-reversible model assumptions35,36. We used Bowker’s test of 201

symmetry to produce subsets of the ‘alphamitoCOGs-24’ dataset by meeting increasingly stringent p-value-202 based thresholds (>0.01, >0.1, >0.3, >0.4 and >0.5, respectively). The Bowker’s test has long been used as 203

an overall test for symmetry36. The test assesses symmetry in an r × r contingency table with the ij-th cell 204 containing the observed frequency nij. The null hypothesis for symmetry is H0 = nij = nji, i ≠ j, i,j = 1,…,r, 205

and the test value is computed by using equation (1). 206

(1) 207

The test statistics follows !2 distribution with the number of degrees of freedom equal to the number of 208

comparisons (nij vs nji) made. 209

The scoring function (SF) utilized for symmetry-based alignment trimming employed here is a sum of 210

absolute values of natural logarithms of Bowker's test's p-values, each raised to a certain power (15 as the 211 default value). SF can be computed as a mean over the values in an upper or lower triangular part of a square 212

matrix which rows and columns represent taxa, populated with |ln p|x values for Bowker’s tests among these 213 taxa, as shown in equation (2). 214

(2) 215

wherein h is the number of taxa in the msa, and pab is a p value for the sequences a and b. 216

The script which performs symmetry-based trimming (available as Supplementary Software) deletes a site 217 in an alignment, computes a SF value and restores the original alignment. The operation is performed for 218

every alignment site. Then, the site which removal results in lowest SF value is deleted irreversibly. The 219 procedure is repeated for each shortened alignment subset until the lowest p-value for a pair-wise Bowker’s 220

test in the trimmed dataset exceeds certain p-value-based threshold(s). 221

Exponentiation in formula 2 leads to a sooner recovery of trimmed subsets. The exponentiation 222 disproportionally increases the addend values in formula 2 (|ln pab|x) for smaller p values. For instance, the 223

default addend in the formula 2 for p-value 0.5 is 0.004 and the addend for p-value 0.005 is 72789633288. 224 Thus, when there is a disparity in individual p-values in the data, which is the case when the method is 225

needed, the exponentiation increases the relative contribution of the lowest p-values onto the SF value size. 226 At each trimming step the heuristic algorithm identifies a site which removal is likely to improve the worst 227

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(lowest) p-values. The script outputs a trimmed subset when the lowest p-value exceeds the threshold value. 228 The suggested exponentiation, causing preferential improvement of the worst p-values at each site stripping 229 step, is able to deliver a result when less positions are removed. The default exponent value (x = 15) has 230

been determined experimentally. 231

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Functional annotation and phylogenetic reconstruction of the pyruvate oxidation and hydrogen 233 generation enzymes. A subset of taxa in the ‘24-alphamitoCOGs’ dataset with high genome quality were 234

selected based on validation by using CheckM (v1.0.11)37 followed by two filtering criteria: (1) for each 235 composite bin, the original bin with the highest genome completeness were selected; and (2) only 236

bins/genomes with completeness > 80% were kept. Proteomes of the selected 65 genomes/bins were 237 searched against to the KEGG database by using KofamKOALA ‘-E 0.01’ (v1.2.0)38. Hits to the entries 238

K00161, K00169, K00174, K00179 and K18005 were calculated for each taxon (Supplementary Table 7). 239 Proteins annotated as K00161, K00174 and K00179 were aligned by using MUSCLE (v3.8)39 and then 240

trimmed by using trimAl ‘-gappyout’ (v1.4)40, respectively. Maximum-likelihood trees for the trimmed 241 alignments were reconstructed by using IQ-TREE (v1.6.12)17 with the ‘LG+C60+F’ model set. 242

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Supplementary Figures 245

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Supplementary Figure 1 | Bayesian phylogenetic tree of the untreated ‘24-alphamitoCOGs’ dataset. 248 The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, 249 mitochondria. 250 251

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Supplementary Figure 2 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset treated by 253 site exclusion based on the Stuart’s test score. The tree is rooted to the outgroup. Posterior probability 254 support values at nodes are shown. mito, mitochondria. 255 256

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Supplementary Figure 3 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 5% sites 258 excluded based on the !2-score method. The tree is rooted to the outgroup. Posterior probability support 259 values at nodes are shown. mito, mitochondria. 260

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Supplementary Figure 4 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 10% 262 sites excluded based on the !2-score method. The tree is rooted to the outgroup. Posterior probability 263 support values at nodes are shown. mito, mitochondria. 264 265

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Supplementary Figure 5 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 20% 267 sites excluded based on the !2-score method. The tree is rooted to the outgroup. Posterior probability 268 support values at nodes are shown. mito, mitochondria. 269

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Supplementary Figure 6 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 40% 271 sites excluded based on the !2-score method. The tree is rooted to the outgroup. Posterior probability 272 support values at nodes are shown. mito, mitochondria. 273 274

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275 Supplementary Figure 7 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 60% 276 sites excluded based on the !2-score method. The tree is rooted to the outgroup. Posterior probability 277 support values at nodes are shown. mito, mitochondria. 278 279

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Supplementary Figure 8 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 5% sites 281 excluded based on the evolving-rate score method. The tree is rooted to the outgroup. Posterior probability 282 support values at nodes are shown. mito, mitochondria. 283 284

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Supplementary Figure 9 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 10% 286 sites excluded based on the evolving-rate score method. The tree is rooted to the outgroup. Posterior 287 probability support values at nodes are shown. mito, mitochondria. 288

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289 Supplementary Figure 10 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 20% 290 sites excluded based on the evolving-rate score method. The tree is rooted to the outgroup. Posterior 291 probability support values at nodes are shown. mito, mitochondria. 292 293

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294 Supplementary Figure 11 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 40% 295 sites excluded based on the evolving-rate score method. The tree is rooted to the outgroup. Posterior 296 probability support values at nodes are shown. mito, mitochondria. 297 298

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Supplementary Figure 12 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 60% 300 sites excluded based on the evolving-rate score method. The tree is rooted to the outgroup. Posterior 301 probability support values at nodes are shown. mito, mitochondria. 302 303

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Supplementary Figure 13 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 5% 305 sites excluded based on the ɀ-score method. The tree is rooted to the outgroup. Posterior probability 306 support values at nodes are shown. mito, mitochondria. 307 308

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324 Supplementary Figure 17 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset with 60% 325 sites excluded based on the ɀ-score method. The tree is rooted to the outgroup. Posterior probability 326 support values at nodes are shown. mito, mitochondria. 327 328

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Supplementary Figure 18 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset treated by 330 site exclusion based on the Bowker’s test score method with P-value > 0.01. The tree is rooted to the 331 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. 332 333

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334 Supplementary Figure 19 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset treated by 335 site exclusion based on the Bowker’s test score method with P-value > 0.1. The tree is rooted to the 336 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. 337 338

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339 Supplementary Figure 20 | Bayesian phylogenetic tree of the ‘24-alphamitoCOGs’ dataset treated by 340 site exclusion based on the Bowker’s test score method P-value > 0.3. The tree is rooted to the outgroup. 341 Posterior probability support values at nodes are shown. mito, mitochondria. 342 343

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Supplementary Figure 23 | Relationships between alignment sites, the phylogenetic position of 356 mitochondria and model fit (max heterogeneity across taxa test) based on different datasets, site-357 exclusion and taxon-selection approaches. Bayesian inference with model CAT+GTR was conducted. X 358 axel shows the number of sites in each dataset for phylogenetic inference. Y axle shows the Z-scores of the 359 ‘max heterogeneity across taxa’ posterior predictive test. Numbers aside markers show node support values 360 (posterior probability support values) of the consensus trees. Values ⩾95 are in bold. Strings in parentheses 361 show the closest relatives of mitochondria in the tree. R, Rickettsiales. T, Tistrella mobilis. F2, FEMAG II. 362 AII, Alpha II. AIII, Alpha III. GT, Geminicoccus roseus and T. mobilis. MA9, MarineAlpha9. mito-in, 363 mitochondria branch within Alphaproteobacteria. mito-out, mitochondria branch outside 364 Alphaproteobacteria. mito-in AlphaIIb, mitochondria brance within the AlphaIIb clade of 365 Alphaproteobacteria. a, site-exclusion methods applied on the ‘24-alphamitoCOGs’ dataset in Martijn et al. 366 (2018). Trees are shown in Supplementary Figure 1–22. b, !2-score based site-exclusion and taxon-367 reduction methods applied on the subsets of the ‘Modified24’ and the ‘Modified18’ datasets, respectively, 368 containing only the backbone, Rickettsiales and mitochondrial sequences. Trees are shown in 369 Supplementary Figure 35, 37–42. c, !2-score based site-exclusion and taxon-reduction methods applied on 370 the subsets of the ‘Modified24’ and the ‘Modified18’ datasets, respectively, containing only the backbone, 371 FEMAGs and mitochondrial sequences. Trees are shown in Supplementary Figure 36, 42–48. d, !2-score 372 based site exclusion applied on the subsets of the ‘Modified24-AlphaII’, the ‘Modified24-AlphaII-MoreTaxa’ 373 and the ‘Modified18-AlphaII’ datasets, respectively. Trees are shown in Fig. 4 and Supplementary Figure 374 50–58. 375 376

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Supplementary Figure 24 | GC content and amino acid compositional heterogeneity among 380 alphaproteobacterial lineages and mitochondria. Dots represent taxa. Lineages are colored according to 381 Fig. 3 except empty dots represent Beta-, Gammaproteobacteria and Magnetococcales. a, taxa in the 382 ‘Modified24’ dataset. b, taxa in the ‘Modified18’ dataset. 383 384

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Supplementary Figure 25 | Bayesian phylogenetic tree of the slowly-evolving backbone taxa of Alphaproteobacteria. The tree is rooted to the outgroup. Posterior 387 probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 388 389

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Supplementary Figure 26 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria and Holosporales. The tree is rooted to the outgroup. Posterior 391 probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 392 393

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Supplementary Figure 27| Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria and Pelagibacterales. The tree is rooted to the outgroup. Posterior 395 probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 396 397

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Supplementary Figure 28 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria and alpha proteobacterium HIMB59. The tree is rooted to the 399 outgroup. Posterior probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 400 401

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Supplementary Figure 29 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria and Rickettsiales. The tree is rooted to the outgroup. Posterior 404 probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 405 406

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Supplementary Figure 30 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria and FEMAGs. The tree is rooted to the outgroup. Posterior 408 probability support values at nodes are shown. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 409 410

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Supplementary Figure 31 | Bayesian phylogenetic tree of the slowly-evolving backbone taxa of Alphaproteobacteria and mitochondria. The tree is rooted to the 412 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 413

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Supplementary Figure 32 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, Holosporales and mitochondria. The tree is rooted to the 415 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 416

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Supplementary Figure 33 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, Pelagibacterales and mitochondria. The tree is rooted to the 418 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 419

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Supplementary Figure 34 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, alpha proteobacterium HIMB59 and mitochondria. The tree 421 is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ 422 dataset. 423

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Supplementary Figure 35 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, Rickettsiales and mitochondria. The tree is rooted to the 425 outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 426

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Supplementary Figure 36 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, FEMAGs and mitochondria. The tree is rooted to the outgroup. 428 Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 429

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Supplementary Figure 37 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, Rickettsiales and mitochondria after 5% sites excluded. The 431 !2-score method for site exclusion was applied. The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on 432 the ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 433

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Supplementary Figure 42 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, Rickettsiales and mitochondria after taxon reduction. The !2-451 score method for site exclusion was applied. The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the 452 ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 453

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Supplementary Figure 43 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, FEMAGs and mitochondria after 5% sites excluded. The !2-455 score method for site exclusion was applied. The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the 456 ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 457 458

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Supplementary Figure 44 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, FEMAGs and mitochondria after 10% sites excluded. The !2-460 score method for site exclusion was applied. The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the 461 ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 462

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Supplementary Figure 48 | Bayesian phylogenetic tree of the backbone taxa of Alphaproteobacteria, FEMAGs and mitochondria after taxon reduction. The !2-476 score method for site exclusion was applied. The tree is rooted to the outgroup. Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the 477 ‘Modified24’ dataset. b, based on the ‘Modified18’ dataset. 478 479

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Supplementary Figure 49 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria. The tree is rooted to the outgroup (Alpha IIa). Posterior 481 probability support values at nodes are shown. a, based on the ‘Modified24-AlphaII’ dataset. b, based on the ‘Modified18-AlphaII’ dataset. 482 483

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Supplementary Figure 50 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria and mitochondria. The tree is rooted to the outgroup (Alpha 486 IIa). Posterior probability support values at nodes are shown. mito, mitochondria. a, based on the ‘Modified24-AlphaII’ dataset. b, based on the ‘Modified18-AlphaII’ dataset. 487 488

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Supplementary Figure 51 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria and mitochondria after 5% sites excluded. The !2-score 491 method for site exclusion was applied. The tree is rooted to the outgroup (Alpha IIa). Posterior probability support values at nodes are shown. mito, mitochondria. a, based 492 on the ‘Modified24-AlphaII’ dataset. b, based on the ‘Modified18-AlphaII’ dataset. 493 494

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Supplementary Figure 56 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria and mitochondria after 5-10% sites excluded based on the 521 ‘Modified24-AlphaII-MoreTaxa’ dataset. The !2-score method for site exclusion was applied. The tree is rooted to the outgroup (Alpha IIa). Posterior probability support 522 values at nodes are shown. mito, mitochondria. a, 5% sites excluded. b, 10% sites excluded. 523 524

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Supplementary Figure 57 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria and mitochondria after 20-40% sites excluded based on 527 the ‘Modified24-AlphaII-MoreTaxa’ dataset. The !2-score method for site exclusion was applied. The tree is rooted to the outgroup (Alpha IIa). Posterior probability 528 support values at nodes are shown. mito, mitochondria. a, 20% sites excluded. b, 40% sites excluded. 529 530

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Supplementary Figure 58 | Bayesian phylogenetic tree of the Alpha II subclade of Alphaproteobacteria and mitochondria after 60% sites excluded based on the 533 ‘Modified24-AlphaII-MoreTaxa’ dataset. The !2-score method for site exclusion was applied. The tree is rooted to the outgroup (Alpha IIa). Posterior probability support 534 values at nodes are shown. mito, mitochondria. 535

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538 Supplementary Figure 59 | Maximum-likelihood phylogenetic tree of pyruvate dehydrogenase E1 539 component alpha subunit (PdhA) found in the taxa of the ‘24-alphamitoCOGs’ dataset. The tree is 540 rooted to the midpoint. Node values show the ultra-fast bootstraps based on 1,000 iterations. Taxa are541 assignedtosubcladesnamedaccordingtoSupplementary Table 3. 542

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Supplementary Figure 60 | Maximum-likelihood phylogenetic tree of pyruvate ferredoxin oxidoreductase found in the taxa of the ‘24-alphamitoCOGs’ dataset. 546 The trees are rooted to the midpoints. Node values show the ultra-fast bootstraps based on 1,000 iterations. Taxa are assigned to subclades named according to547 Supplementary Table 3. a, 2-oxoglutarate/2-oxoacid ferredoxin oxidoreductase subunit alpha (OorA). b, indolepyruvate ferredoxin oxidoreductase subunit alpha (IorA). 548

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