A phylometagenomic exploration of oceanic alphaproteobacteria reveals mitochondrial relatives unrelated to the SAR11 clade

Abstract

Background: According to the endosymbiont hypothesis, the mitochondrial system for aerobic respiration was derived from an ancestral Alphaproteobacterium. Phylogenetic studies indicate that the mitochondrial ancestor is most closely related to the Rickettsiales. Recently, it was suggested that Candidatus Pelagibacter ubique, a member of the SAR11 clade that is highly abundant in the oceans, is a sister taxon to the mitochondrial-Rickettsiales clade. The availability of ocean metagenome data substantially increases the sampling of Alphaproteobacteria inhabiting the oxygen-containing waters of the oceans that likely resemble the originating environment of mitochondria.

Methodology/principal findings: We present a phylogenetic study of the origin of mitochondria that incorporates metagenome data from the Global Ocean Sampling (GOS) expedition. We identify mitochondrially related sequences in the GOS dataset that represent a rare group of Alphaproteobacteria, designated OMAC (Oceanic Mitochondria Affiliated Clade) as the closest free-living relatives to mitochondria in the oceans. In addition, our analyses reject the hypothesis that the mitochondrial system for aerobic respiration is affiliated with that of the SAR11 clade.

Conclusions/significance: Our results allude to the existence of an alphaproteobacterial clade in the oxygen-rich surface waters of the oceans that represents the closest free-living relative to mitochondria identified thus far. In addition, our findings underscore the importance of expanding the taxonomic diversity in phylogenetic analyses beyond that represented by cultivated bacteria to study the origin of mitochondria.

Figure 1. Schematic overview of data selection procedure.

The flow scheme depicted here displays which datasets have been used and how they were analysed (for more details, see Materials and Methods). Three different datasets have been used, being the COG database (taken from STRING ; “COG”, orange shading), the GOS database (“GOS”, blue shading), and a local database of proteins that are encoded by mitochondrial genomes (‘MT’, green shading). First, homologs were retrieved for each of the 67 proteins encoded by the R. americana mitochondrial genome for each of these datasets using BlastP searches. Next, paralogs and distant homologs were removed from the retrieved GOS and MT hits by performing BlastP searches against the COG database and using stringent cut-off filters. Since the amounts of retrieved GOS homologs was too high for Bayesian analyses, two strategies were used for down-sampling: One approach involved a pruning step in which the amount of GOS homologs was reduced while reducing the phylogenetic diversity, another approach involved the targeted sub-sampling of GOS sequences that were placed as a neighbour to the mitochondrial clade in a jack-knifing screen (see Material and Methods for details). Then, the MT and COG datasets were combined and subjected to phylogenetic analysis (PhyML), selecting only those proteins whose evolutionary history was evolutionary coherent (i.e. Alphaproteobacteria formed one clade, and mitochondria formed one clade). The resulting protein datasets are referred to as the ‘reference datasets’. The reference datasets were used for three independent analyses: (i) Proteins of the reference dataset were concatenated and subjected to Bayesian analysis; Proteins of the reference dataset were either combined with the pruned (ii) or sub-sampled (iii) GOS datasets, followed by Bayesian analysis.

Figure 2. The taxonomic sampling of Alphaproteobacteria is enhanced by the addition of GOS sequences.

A phylogenetic analysis of 1641 GOS RPS2 homologs reveals that the taxonomic sampling is increased for most major bacterial clades, and specifically for the Alphaproteobacteria. The vast majority (82.5%) of the extracted RPS2 homologs that are placed in the Alphaproteobacteria is associated with the SAR11 clade, which includes Ca. Pelagibacter ubique. Rhodobacterales, Rhodospirillales, Rhizobiales and Rickettsiales are associated with 9.3, 7.4, 0.6 and 0.2% of the GOS sequences, respectively. The sequence data covered by the GOS dataset greatly increases the previous sampling of the SAR11-clade, as well as of other oceanic bacteria that lack sequenced representatives, thereby representing a rich source of sequences useful for examining the placement of mitochondria in relation to bacteria adapted to the upper surface waters of the oceans. For clarity, all GOS clades have been collapsed. Annotation of collapsed GOS clades is as follows: l and n represent the total branch length and the number of GOS sequences within collapsed GOS clades, respectively, and shading is according to the total branch length of the clade as indicated.

Figure 3. Phylogenetic analysis COX1 orthologs extracted from the GOS database.

A phylogenetic tree is shown that is based on an alignment of COX1 protein sequences from the reference set of alphaproteobacterial and mitochondrial species supplemented with a pruned set of GOS sequences (shown in bold). The main alphaproteobacterial orders Rickettsiales, Rhodobacteriales, Rhodospirillales, Sphingomonadales, Caulobacteriales and Rhizobiales are indicated in coloured shading. Note that some GOS sequences are placed close to or at the root of the Rickettsiales clade and that the SAR11 clade encompassing Ca. Pelagibacter ubique is unrelated to the mitochondrial lineage. The tentative placement of the outgroup (OG) is indicated with an arrow. Phylogenies were produced using Bayesian methods with the CAT model. Numbers at nodes denote posterior probability values. Numbers associated with GOS clades denote the number of GOS sequences here represented as a single terminal node.

Figure 4. Phylogenetic analysis COB orthologs extracted from the GOS database.

A phylogenetic tree is shown that is based on an alignment of COB protein sequences from the reference set of alphaproteobacterial and mitochondrial species supplemented with a pruned set of GOS sequences (shown in bold). The main alphaproteobacterial orders Rickettsiales, Rhodobacteriales, Rhodospirillales, Sphingomonadales, Caulobacteriales and Rhizobiales are indicated in coloured shading. Note that the SAR11 clade encompassing Ca. Pelagibacter ubique is unrelated to the mitochondrial lineage. The tentative placement of the outgroup (OG) is indicated with an arrow. Phylogenies were produced using Bayesian methods with the CAT model. Numbers at nodes denote posterior probability values. Numbers associated with GOS clades denote the number of GOS sequences here represented as a single terminal node.

Figure 5. Phylogenetic analysis NAD7 orthologs extracted from the GOS database.

A phylogenetic inference based on an alignment of NAD7 protein sequences from the reference set of alphaproteobacterial and mitochondrial species supplemented with a pruned set of GOS sequences (shown in bold). The main alphaproteobacterial orders Rickettsiales, Rhodobacteriales, Rhodospirillales, Sphingomonadales, Caulobacteriales and Rhizobiales are indicated. Note that some GOS sequences are placed at the root of the Rickettsiales clade and that the SAR11 clade encompassing Ca. Pelagibacter ubique is unrelated to the mitochondrial lineage. The tentative placement of the outgroup (OG) is indicated with an arrow. Phylogenies were produced using Bayesian methods with the CAT model. Numbers at nodes denote posterior-probability values. Numbers associated with GOS clades denote the number of GOS sequences here represented as a single terminal node.

Figure 6. Deeply diverging alphaproteobacterial GOS sequences in a COX1–COX2 protein tree.

A systematic search for GOS sequences was performed to identify alphaproteobacterial clades that could potentially represent the closest free-living ancestor of the mitochondria. The Bayesian phylogenetic analysis of concatenated COX1–COX2 sequences encoded by GOS scaffolds revealed a deeply diverging clade of GOS sequences that are associated with the Rickettsiales (pp = 0.99). The tentative placement of the outgroup (OG) is indicated with an arrow. Phylogenies were produced using Bayesian methods with the CAT model. Numbers at nodes denote posterior-probability values.

Figure 7. Ca. Pelagibacter ubique is placed with free-living Alphaproteobacteria in a phylogenetic tree inferred from concatenated protein sequences.

A phylogenetic inference of the alphaproteobacterial tree topology based on a concatenated alignment of 42 mitochondrially encoded proteins reveals a well-supported alphaproteobacterial phylogeny, in which the SAR11 clade, here represented by Ca. Pelagibacter ubique is placed among the free-living alphaproteobacteria rather than with the Rickettsiales. The tentative placement of the outgroup (OG) is indicated with an arrow. Phylogenies were produced using Bayesian methods with the CAT model. Posterior-probability values of 1 are not shown.