Endosymbiosis and the Eukaryotic Cell

Gray, Michael W.  “Mitochondrial Evolution.”  Cold Spring Harbor Perspectives in Biology 4(9):a011403.  2012.  A review by a Dalhousie University biochemist whose group sequenced many of the protist mitochondrial genomes discussed here.  It sets the bacterial features of mitochondrial genomes beside surveys of the mitochondrial proteome, which show that only a minority of mitochondrial proteins trace clearly to alphaproteobacteria.  Mirror:  PMC

1. 1a.

   Section Abstract:  “Viewed through the lens of the genome it contains, the mitochondrion is of unquestioned bacterial ancestry, originating from within the bacterial phylum α-Proteobacteria (Alphaproteobacteria).” ↩

2. 1b.

   Section What do genetic, genomic, and phylogenomic data tell us:  “Second, in plant and many ancestral protist mtDNAs, ribosomal protein genes are clustered in the same transcriptional order in which they appear in the corresponding bacterial operons.” ↩

3. 1c.

   Section Origin and evolution of the mitochondrial proteome:  “Considering the clear α-Proteobacterial signature of the mitochondrial genome, we might have anticipated that the mitochondrial proteome would consist largely of α-Proteobacteria-like proteins plus proteins exclusive to the eukaryotic domain, “invented” during the evolutionary transition from bacterial endosymbiont to organelle. Somewhat surprisingly, this expectation has not been met. In surveys of the yeast mitochondrial proteome ( Karlberg et al. 2000 ; Marcotte et al. 2000 ), only ∼10%–15%—a much smaller proportion than might have been anticipated—proved to originate clearly from the α-Proteobacterial lineage.” ↩

Kay, Christopher J. et al.  “Dated gene duplications elucidate the evolutionary assembly of eukaryotes.”  Nature 650:129–140.  2025.  A molecular-clock study that dates gene duplications made before the last common ancestor of eukaryotes, using a relaxed clock with 18 fossil calibrations, to put the steps of eukaryote origin in order.  It concludes that the host had a nucleus and internal membranes before it took in the mitochondrion.  The dates are model outputs.  Mirror:  PMC

1. 2a.

   Section Abstract:  “there are no living lineages representative of intermediate steps within eukaryogenesis.” ↩ ↩

2. 2b.

   Section The development of key eukaryotic traits:  “Many of the key components of the nucleus, such as nuclear pores, appear to be eukaryote-specific innovations that lack homologues in modern prokaryotes.” ↩

3. 2c.

   Section Dating mitochondrial endosymbiosis:  “the eukaryotic host cell already had complex cellular features before mitochondrial endosymbiosis, including an elaborated cytoskeleton, membrane trafficking, endomembrane, phagocytotic machinery and a nucleus, all between 3.0 and 2.25 billion years ago, after which mitochondrial endosymbiosis occurred.” ↩

Roger, Andrew J. et al.  “The Origin and Diversification of Mitochondria.”  Current Biology 27(21):R1177–R1192.  2017.  A review by three cell biologists of what the mitochondrion's origin required and of how mitochondria later diversified.  It is used here for the mainstream's own list of the changes the transition needed.

1. 3a.

   Section Abstract:  “The transition from endosymbiotic bacterium to permanent organelle entailed a massive number of evolutionary changes including the origins of hundreds of new genes and a protein import system, insertion of membrane transporters, integration of metabolism and reproduction, genome reduction, endosymbiotic gene transfer, lateral gene transfer and the retargeting of proteins. These changes occurred incrementally as the endosymbiont and the host became integrated.” ↩ ↩

Criswell, Daniel.  “A Review of Mitoribosome Structure and Function Does not Support the Serial Endosymbiotic Theory.”  Answers Research Journal 2:107–115.  2009.  A young-earth review in Answers in Genesis's research journal.  It compares mammalian mitochondrial ribosomes with bacterial ribosomes by size, RNA content, protein content and structure, and argues that the differences rule out descent.

1. 4a.

   Section Abstract:  “Mitochondria have a few minor characteristics that are similar to bacteria, such as their size, lack of introns in the mitochondrial genome, and a bi-layer cell covering that have been used to support the theory of serial endosymbiosis. However, there are also significant differences that make the transition of a bacterium into a eukaryotic mitochondrion impossible.” ↩

2. 4b.

   Section Abstract:  “It is apparent from the knowledge gained about mitochondria ribosome structure and function since the proposal of the Serial Endosymbiosis Theory that prokaryotes are not the ancestors of eukaryote or mammalian mitochondria.” ↩

3. 4c.

   Section Introduction:  “Reclinomonas americana mitochondria have the most protein coding genes (67) of any known eukaryote mitochondrial DNA”. ↩

4. 4d.

   Section Abstract:  “However, the structure of mammalian mitochondrial ribosomes and their RNA and amino acid sequences indicate that mammalian mitochondrial ribosomes are completely different from prokaryotic ribosomes.” ↩

Tan, Change, and Jeffrey P. Tomkins.  “Information Processing Differences Between Archaea and Eukarya—Implications for Homologs and the Myth of Eukaryogenesis.”  Answers Research Journal 8:121–141.  2015.  A young-earth review, the first of a pair, by a University of Missouri biologist and an Institute for Creation Research geneticist.  It lists differences between the DNA copying, gene reading and protein-making machinery of archaea and eukaryotes and argues that each is a gap evolution cannot cross.  It does not discuss the Asgard archaea.

1. 5a.

   Page 130:  “In fact, it has recently been found that mitochondrial ribosomes are very different from those of bacteria or archaea or eukarya”.  Page 121:  “These selected discontinuous molecular chasms highlight the impossibility for eukarya having evolved from archaea.” ↩ ↩

2. 5b.

   Page 130:  “Eukarya also contain many (more than 100) mitochondrial-specific ribosomal proteins, which do not have any homologs in bacteria or archaea (Amunts et al. 2014; Desmond et al. 2011; Greber et al. 2014; Rackham and Filipovska 2014; Zikova et al. 2008).” ↩

Tomkins, Jeffrey P.  “Endosymbiosis: A Theory in Crisis.”  Acts & Facts 44(11).  2015.  A short magazine article by an Institute for Creation Research geneticist.  It argues that genome data have thrown endosymbiosis "into utter confusion," resting mainly on Michael Gray's 2015 finding that only 10 to 20% of mitochondrial proteins carry a clear alphaproteobacterial signal.

1. 6a.

   Second paragraph:  “This functional combination looks similar to how bacterial DNA is constructed and how bacteria reproduce, but the appearances are only superficial.” ↩

2. 6b.

   Paragraph beginning "The sequences of many eukaryotic genomes":  “these correspondences are plausibly explained by the standard engineering concept of code re-use”. ↩

3. 6c.

   Paragraph after the quotation from Gray:  “This lack of evidence for mitochondrial genes derived from bacterial origin”.  “is a serious problem for the evolutionary story.” ↩

4. 6d.

   Third paragraph:  “invoking a magical jump in lieu of any scientific explanation”. ↩

Demick, David.  “Mitochondria—created to energize us.”  Journal of Creation 20(3):10–12.  2006.  A short young-earth article in Creation Ministries International's journal.  It accepts some similarities between mitochondria and bacteria, argues that they are selected and do not prove kinship, and offers design for similar jobs as the better reading.

1. 7a.

   Section Conclusion:  “The creationist model, holding that structures may look similar because they were designed to do similar jobs, is a more reasonable way to view the miracle of mitochondria.” ↩

2. 7b.

   Section Intracellular control:  “Yet, nothing really like the formation of mitochondria has ever been observed.” ↩ ↩

3. 7c.

   Section Mitochondrial DNA:  “For example, human mitochondria have 83 proteins, but only 13 are coded by mtDNA”. ↩

Purdom, Georgia.  “"Non-Evolution" of the Appearance of Mitochondria and Plastids in Eukaryotes: Challenges to Endosymbiotic Theory.”  Answers in Genesis.  Oct 11, 2006.  A young-earth web article by Georgia Purdom of Answers in Genesis.  It grants the organelles' bacterial features, then argues that gene transfer to the nucleus would strand the organelle unless a protein-import route already existed, and that no organism shows the intermediate stages.

1. 8a.

   Opening section:  “These organelles share some characteristics with bacteria/cyanobacteria, including circular DNA, division by binary fission, and membrane and ribosome similarities.”  Section Conclusion:  “Again, the question can be asked, common ancestor or common designer?” ↩

2. 8b.

   Section Complex Relationships Between Cells and Organelles:  “If there is no way to form these pathways, then the organelles would become obsolete once they started transferring their genes to the nucleus.” ↩ ↩

3. 8c.

   Paragraph on the proposed stages of plastid endosymbiosis:  “In a classic case of circular reasoning-they developed the stages after studying the organisms!” ↩

Rana, Fazale.  “Can a Creation Model Explain the Origin of Mitochondria?”  Reasons to Believe.  Nov 23, 2016.  A blog essay by Fazale Rana of Reasons to Believe, an old-earth creationist ministry.  It accepts the evidence usually given for the endosymbiont hypothesis and argues that the shared features, especially the lipid cardiolipin, have functional reasons that a designer would also use.

1. 9a.

   Section A Creation Model Perspective on Mitochondria:  “I maintain that it is reasonable to view eukaryotic cells as the work of a Creator, with the shared similarities between mitochondria and bacteria reflecting common design rather than common descent.” ↩

Mitchell, Elizabeth.  “Just Endosymbiosis.”  Answers in Genesis.  Jan 21, 2012.  A young-earth news commentary on the Answers in Genesis site about a ciliate that keeps captured algae.  It argues that such cases are ordinary symbiosis, that gene transfer and protein import could not be built step by step, and that no transitional forms exist.

1. 10a.

   Paragraph listing the problems of endosymbiotic theory:  “Furthermore, no transitional forms have ever been found.” ↩

McLatchie, Jonathan.  “Answering Farina on Irreducible Complexity.”  Evolution News, Discovery Institute.  Jun 22, 2023.  A reply on the Discovery Institute's news site to a video by the science YouTuber Dave Farina.  One paragraph answers Farina's example of sea slugs that keep stolen chloroplasts, accepting the 2008 report of gene transfer that Farina cited.

1. 11a.

   Paragraph beginning "As a further example":  “The paper determines that the essential plastid proteins are supplied by the sea slug itself, and that the genes which support photosynthesis have been acquired through horizontal gene transfer. But this does not involve the evolution of any new complex traits.” ↩ ↩

Lang, B. Franz et al.  “An ancestral mitochondrial DNA resembling a eubacterial genome in miniature.”  Nature 387(6632):493–497.  1997.  The complete sequence of the mitochondrial genome of the freshwater protist Reclinomonas americana, which carries more genes than any mitochondrial genome known at the time, including genes for a bacterial-type RNA polymerase not found in other mitochondria.

1. 12a.

   Section Abstract:  “R. americana mtDNA (69,034 base pairs) contains the largest collection of genes (97) so far identified in any mtDNA, including genes for 5S ribosomal RNA, the RNA component of RNase P, and at least 18 proteins not previously known to be encoded in mitochondria. Most surprising are four genes specifying a multisubunit, eubacterial-type RNA polymerase.” ↩

Burger, Gertraud et al.  “Strikingly Bacteria-Like and Gene-Rich Mitochondrial Genomes throughout Jakobid Protists.”  Genome Biology and Evolution 5(2):418–438.  2013.  A comparison of nine mitochondrial genomes from five genera of jakobid protists.  It finds the bacterial features first seen in Reclinomonas across the whole group, including operon-like gene order closer to free-living alphaproteobacteria than to the parasitic Rickettsiales, and one genome with even more genes.  Mirror:  PMC

1. 13a.

   Section Abstract:  “This genome also encodes the largest known gene set among mitochondrial DNAs (mtDNAs), including the RNA subunit of RNase P (transfer RNA processing), a reduced form of transfer-messenger RNA (translational control), and a four-subunit bacteria-like RNA polymerase, which in other eukaryotes is substituted by a nucleus-encoded, single-subunit, phage-like enzyme. Further, protein-coding genes are preceded by potential Shine–Dalgarno translation initiation motifs.” ↩ ↩

2. 13b.

   Section Abstract:  “Similarly, a long, highly conserved gene cluster in jakobid mtDNAs, which is a clear vestige of prokaryotic operons, displays a gene order more closely resembling that in free-living α-proteobacteria than in Rickettsiales species.” ↩

Petrov, Anton S. et al.  “Structural Patching Fosters Divergence of Mitochondrial Ribosomes.”  Molecular Biology and Evolution 36(2):207–219.  2019.  A comparison of mitochondrial ribosome structures and RNA across many species, which maps where each lineage lost or gained RNA and protein.  It proposes that lost RNA was "patched" by proteins the cell already had, a mechanism inferred from the pattern rather than watched.  Mirror:  PMC

1. 14a.

   Section Abstract:  “Unlike other ribosomes, mitoribosomes are highly variable across species. The basis for this diversity is not known.” ↩

Waltz, Florent et al.  “Small is big in Arabidopsis mitochondrial ribosome.”  Nature Plants 5(1):106–117.  2019.  A biochemical and cryo-electron-microscope study of the mitochondrial ribosome of the plant Arabidopsis, which found 19 plant-specific proteins and an unusually large small subunit.

1. 15a.

   Section Abstract:  “Overall, our results show that Arabidopsis mitoribosomes are substantially divergent from bacterial and other eukaryote mitoribosomes, in terms of both structure and protein content.” ↩

Amunts, Alexey et al.  “Structure of the Yeast Mitochondrial Large Ribosomal Subunit.”  Science 343(6178):1485–1489.  2014.  A near-atomic cryo-electron-microscope structure of the large subunit of the yeast mitochondrial ribosome.  It is one of the papers Tan and Tomkins cite, and it counts which of the subunit's proteins have bacterial relatives.

1. 16a.

   Section Mitochondria-specific protein elements:  “Of the 39 proteins built into the 54S, 26 share homology with bacterial ribosomes, 8 are present in both yeast and mammalian mitochondria and 5 are unique to yeast mitochondria”. ↩

Bieri, Philipp et al.  “The complete structure of the chloroplast 70S ribosome in complex with translation factor pY.”  The EMBO Journal 36(4):475–486.  2017.  A 3.4-ångström cryo-electron-microscope structure of the spinach chloroplast ribosome from the ETH Zurich laboratory of Nenad Ban.  It compares the chloroplast ribosome part by part with the bacterial ribosome.  Mirror:  PMC

1. 17a.

   Section Introduction:  “The chloroplast ribosomal RNA (rRNA) (total 4,524 nucleotides in spinach) is about the same length as in the bacterial ribosome (total 4,566 nucleotides in Escherichia coli ).”  Section Results and Discussion:  “In contrast to the solvent side, the rRNA core and the interface of the two subunits are structurally much more conserved relative to the bacterial ribosome”. ↩

Hobbie, Sven N. et al.  “Genetic analysis of interactions with eukaryotic rRNA identify the mitoribosome as target in aminoglycoside ototoxicity.”  Proceedings of the National Academy of Sciences 105(52):20888–20893.  2008.  A laboratory study that built bacterial ribosomes carrying the human mitochondrial or cytosolic drug-binding site and tested antibiotics against them.  The mitochondrial versions were hit by aminoglycosides in proportion to the drugs' damage to hearing.  Mirror:  PMC

1. 18a.

   Section Abstract:  “Sequence alterations that correspond to the mitochondrial deafness mutations A1555G and C1494T increased drug-binding and rendered the ribosomal decoding site hypersusceptible to aminoglycoside-induced mistranslation and inhibition of protein synthesis.” ↩

Hirano, Takayuki et al.  “Moss Chloroplasts Are Surrounded by a Peptidoglycan Wall Containing D-Amino Acids.”  The Plant Cell 28(7):1521–1532.  2016.  A genetic and labelling study in the moss Physcomitrella.  Knocking out a bacterial-type cell-wall gene gave giant chloroplasts, and a clickable D-amino-acid label showed a peptidoglycan wall around every chloroplast.

1. 19a.

   Section Abstract:  “The ∆Pp-ddl line complemented with EDA-DA showed that moss chloroplasts are completely surrounded by peptidoglycan. Our findings strongly suggest that the moss plastids have a peptidoglycan wall containing d-amino acids. By contrast, no plastid phenotypes were observed in the T-DNA tagged ddl mutant lines of Arabidopsis thaliana.” ↩ ↩

Gray, Michael W.  “Mosaic nature of the mitochondrial proteome: Implications for the origin and evolution of mitochondria.”  Proceedings of the National Academy of Sciences 112(33):10133–10138.  2015.  A review for a 2014 National Academy of Sciences colloquium.  It sets out how small a share of the mitochondrial proteome traces clearly to alphaproteobacteria, and weighs where the rest came from.  It is the source Tomkins quotes.  Mirror:  PMC

1. 20a.

   First paragraph of the main text:  “What was not anticipated was how relatively few mitochondrial proteins with bacterial homologs would group specifically with α-Proteobacteria in phylogenetic reconstructions: At most, only 10–20% of any of the mitochondrial proteomes examined so far display a robust α-proteobacterial signal.” ↩

2. 20b.

   First paragraph of the main text:  “That the mitochondrial genome is of bacterial (specifically α-proteobacterial) origin is now indisputable”.  “This minority group includes proteins encoded by the mitochondrial genome as well as nucleus-encoded mitochondrial proteins whose genes we infer were transferred to the nucleus via endosymbiotic gene transfer (EGT) during mitochondrial evolution.” ↩

3. 20c.

   Section Composition of the Mitochondrial Proteome:  “it has become apparent that the LMCA was already as complex in its key functions as modern mitochondria; importantly, for example, it possessed an essentially complete protein-import apparatus”. ↩

Tan, Change, and Jeffrey P. Tomkins.  “Information Processing Differences Between Bacteria and Eukarya—Implications for the Myth of Eukaryogenesis.”  Answers Research Journal 8:143–162.  2015.  The second of the two young-earth reviews by Tan and Tomkins, comparing bacterial and eukaryotic molecular machinery.  Its one passage on mitochondria reads a 2014 phylogenomic count as weak support for an alphaproteobacterial origin.

1. 21a.

   Page 144:  “Furthermore, of the 243 LECA clades with bacterial origin, only 44 (18%) could be traced to a specific bacteria phylum (41 to Alphaproteobacteria, two to Cyanobacteria, and one to Verrucomicrobiae)”. ↩

2. 21b.

   Page 144:  “Therefore, 311 (~42%) out of the 744 phylogenetic trees generated show that eukaryotes are not monophyletic”. ↩

Rochette, Nicolas C. et al.  “Phylogenomic test of the hypotheses for the evolutionary origin of eukaryotes.”  Molecular Biology and Evolution 31(4):832–845.  2014.  A phylogenetic survey of 554 gene families present in the last common ancestor of eukaryotes, asking which prokaryotes each family groups with.  It is the source Tan and Tomkins draw their count from.

1. 22a.

   Section Results:  “Almost all of them (38 out of 41) were involved in core mitochondrial functions such as protein processing (translation, chaperones), respiration (tricarboxylic acid cycle, oxidative phosphorylation, ATP synthase), and Fe-S cluster biosynthesis.” ↩

Martijn, Joran et al.  “Deep mitochondrial origin outside the sampled alphaproteobacteria.”  Nature 557(7703):101–105.  2018.  A phylogenomic study that added twelve new alphaproteobacterial groups from ocean metagenomes and corrected for known tree-building artefacts.  It places the mitochondrion just outside all sampled alphaproteobacteria.

1. 23a.

   Section Abstract:  “Rather, our analyses indicate that mitochondria evolved from a proteobacterial lineage that branched off before the divergence of all sampled alphaproteobacteria.” ↩

Muñoz-Gómez, Sergio A. et al.  “Site-and-branch-heterogeneous analyses of an expanded dataset favour mitochondria as sister to known Alphaproteobacteria.”  Nature Ecology & Evolution 6(3):253–262.  2022.  A phylogenomic study using 108 mitochondrial proteins of alphaproteobacterial origin and a new model that allows protein composition to vary across the tree.  It places the mitochondrion as sister to the known alphaproteobacteria, answering Martijn and colleagues.

1. 24a.

   Section Abstract:  “The consilience of evidence thus suggests that mitochondria are sister to the Alphaproteobacteria to the exclusion of MarineProteo1 and Magnetococcia.” ↩

Jeon, Kwang W.  “Development of Cellular Dependence on Infective Organisms: Micrurgical Studies in Amoebas.”  Science 176(4039):1122–1123.  1972.  A laboratory study that swapped nuclei and cytoplasm between normal amoebae and amoebae of a strain infected with bacteria five years earlier.  Nuclei from the infected strain could no longer work without the bacteria.

1. 25a.

   Section Abstract:  “After 5 years of infection, the infective bacteria that were initially harmful to the host cells became harmless, and the nucleus of the host cell became dependent on the infective organisms for its normal functions.” ↩

Lorch, I. Joan, and Kwang W. Jeon.  “Rapid Induction of Cellular Strain Specificity by Newly Acquired Cytoplasmic Components in Amoebas.”  Science 211(4485):949–951.  1981.  A follow-up on Jeon's infected amoebae, showing how quickly the bacteria changed the host so that its nuclei no longer matched the cytoplasm of the original strain.

1. 26a.

   Section Abstract:  “A new strain-specific character was induced in amoebas by bacterial endosymbionts after only 10 to 15 cell generations of symbiosis.” ↩

Jeon, Kwang W.  “Genetic and physiological interactions in the amoeba-bacteria symbiosis.”  Journal of Eukaryotic Microbiology 51(5):502–508.  2004.  Jeon's review of the infected amoeba strain decades after the infection, summarising how dependent the host had become and how the bacteria alter its gene use.

1. 27a.

   Section Abstract:  “Each xD amoeba contains about 42,000 symbionts within symbiosomes, and established xD amoebae die if their symbionts are removed.” ↩

Jeon, T. J., and Kwang W. Jeon.  “Gene switching in Amoeba proteus caused by endosymbiotic bacteria.”  Journal of Cell Science 117(4):535–543.  2004.  A laboratory study comparing infected and uninfected amoebae, which found that the bacteria switch the host from one version of a housekeeping enzyme gene to another.

1. 28a.

   Section Abstract:  “The expression of amoeba sams genes was switched from sams1 to sams2 as a result of infection with X-bacteria”. ↩

Nowack, Eva C. M., and Arthur R. Grossman.  “Trafficking of protein into the recently established photosynthetic organelles of Paulinella chromatophora.”  Proceedings of the National Academy of Sciences 109(14):5340–5345.  2012.  A laboratory study using antibodies, electron microscopy and radiolabelling to follow three photosystem proteins whose genes moved from the chromatophore to the nucleus.  The proteins are made in the host cytoplasm and carried into the chromatophore, apparently through the Golgi.  Mirror:  PMC

1. 29a.

   Section Introduction:  “that are delineated from the “host cell” cytoplasm by a double envelope membrane and peptidoglycan layer.” ↩

2. 29b.

   Section Abstract:  “After the initial endosymbiotic event, the CR genome was reduced to approximately 30% of its presumed original size and more than 30 expressed genes were transferred from the CR to the amoebal nuclear genome.”  “Here we report biochemical evidence that PsaE, PsaK1, and PsaK2 are synthesized in the amoeba cytoplasm and traffic into CRs, where they assemble with CR-encoded subunits into photosystem I complexes.” ↩ ↩

3. 29c.

   Section Discussion:  “Trafficking does not appear to depend on a cleavable presequence, although our initial data suggest that proteins routed to the CR pass through the Golgi.” ↩

4. 29d.

   Section Discussion:  “With a growing knowledge of endosymbiotic systems, the border between organelles and endosymbionts has been blurred.” ↩

Nowack, Eva C. M. et al.  “Chromatophore Genome Sequence of Paulinella Sheds Light on Acquisition of Photosynthesis by Eukaryotes.”  Current Biology 18(6):410–418.  2008.  The complete genome of the photosynthetic chromatophore of the amoeba Paulinella chromatophora, compared gene by gene with its free-living cyanobacterial relatives.  It found a genome cut to about a quarter of their gene count and missing essential pathways.

1. 30a.

   Section Abstract:  “Compared to Synechococcus WH5701, a free-living relative of the chromatophore, only 26% of the genes were retained.”  “Our data characterize the chromatophore as a photosynthetic entity that is absolutely dependent on its host for growth and survival.” ↩ ↩

Singer, Anna et al.  “Massive Protein Import into the Early-Evolutionary-Stage Photosynthetic Organelle of the Amoeba Paulinella chromatophora.”  Current Biology 27(18):2763–2773.e5.  2017.  A mass-spectrometry survey of the proteins inside isolated Paulinella chromatophores, combined with computer prediction.  It catalogues the host proteins sent into the organelle and asks where their genes came from.

1. 31a.

   Section Abstract:  “By combining experimental data with in silico predictions, we provide a comprehensive catalog of almost 450 nuclear-encoded, chromatophore-targeted proteins. Interestingly, most imported proteins seem to derive from ancestral host genes, suggesting that the re-targeting of nuclear-encoded proteins that resulted from endosymbiotic gene transfers plays only a minor role at the onset of chromatophore integration.” ↩ ↩

2. 31b.

   Section Abstract:  “Surprisingly, upon heterologous expression in a plant cell, the putative chromatophore targeting signal conferred chloroplast localization.” ↩

Delaye, Luis et al.  “How Really Ancient Is Paulinella Chromatophora?”  PLoS Currents Tree of Life 8.  2016.  A molecular-clock estimate of when the photosynthetic Paulinella split from its non-photosynthetic relatives, which sets an upper limit on the chromatophore's age.  The answer changes with the calibrations chosen.  Mirror:  PMC

1. 32a.

   Section Abstract:  “Our time estimates show that depending on the assumptions made to calibrate the molecular clock, P. chromatophora diverged from heterotrophic Paulinella spp. ~ 90 to 140 Myr ago, thus establishing a maximum date for the origin of the chromatophore.” ↩ ↩

Coale, Tyler H. et al.  “Nitrogen-fixing organelle in a marine alga.”  Science 384(6692):217–222.  2024.  An imaging and proteomics study of the nitrogen-fixing cyanobacterium UCYN-A inside the alga Braarudosphaera bigelowii.  It found that the bacterium divides in step with the alga and imports proteins made from the alga's genes, and proposed calling it an organelle.

1. 33a.

   Section Abstract:  “Here we show that UCYN-A has been tightly integrated into algal cell architecture and organellar division and that it imports proteins encoded by the algal genome. These are characteristics of organelles and show that UCYN-A has evolved beyond endosymbiosis and functions as an early evolutionary stage N2-fixing organelle, or "nitroplast."” ↩

Okamoto, Noriko, and Isao Inouye.  “A Secondary Symbiosis in Progress?”  Science 310(5746):287.  2005.  A short report of a flagellate that carries a green alga, inherits it unevenly at division, and is reshaped by it, as is the alga.  The authors ask, with a question mark, whether this is plastid acquisition caught in progress.

1. 34a.

   Section Abstract:  “This type of life cycle, in which endosymbiont integration has only partially converted the host from predator to autotroph, may represent an early stage of plastid acquisition through secondary symbiosis.” ↩

2. 34b.

   Section Abstract:  “We report a protist, which we tentatively refer to as Hatena, that hosts an endosymbiotic green algal partner but inherits it unevenly.” ↩

Michalik, Anna et al.  “Convergent extreme reductive evolution in ancient planthopper symbioses.”  Nature Communications 17:2473.  2026.  A genomic study of the ancient bacteria Sulcia and Vidania, which live inside planthopper insects, using metagenomic data from 149 planthopper species.  It found that Vidania genomes in two planthopper groups evolved separately into genomes of about 50–52 kb (kilobases, thousands of DNA letters), the smallest non-organellar bacterial genomes known, and the authors themselves frame the result as about the bacteria-organelle boundary, not minimal life, because a host supplies everything else.  The article cites the 50 to 52 kilobase Vidania genomes as examples of the smallest natural genomes, which prove nothing about minimal life.

1. 35a.

   Section Abstract:  “Losing many additional cell-function genes places them very close to organelles of symbiotic origin in the level of host dependence, further blurring the bacteria-organelle boundary.” ↩

Bhattacharya, Debashish et al.  “Genome Analysis of Elysia chlorotica Egg DNA Provides No Evidence for Horizontal Gene Transfer into the Germ Line of This Kleptoplastic Mollusc.”  Molecular Biology and Evolution 30(8):1843–1852.  2013.  A genome study of sea-slug eggs that had never touched algae, searched against a new transcriptome of the slug's algal food and against every earlier candidate gene.  Mary Rumpho, senior author of the 2008 gene-transfer report, is the senior author here.  Mirror:  PMC

1. 36a.

   Section Abstract:  “Our comprehensive analyses fail to provide evidence for alga-derived HGT into the germ line of the sea slug.” ↩

2. 36b.

   Section Introduction:  “retains functional, nondividing plastids in the cells lining the digestive tract for several months”.  “naturally aposymbiotic E. chlorotica egg DNA that had never come in contact with algal prey”. ↩

Maeda, Taro et al.  “Chloroplast acquisition without the gene transfer in kleptoplastic sea slugs, Plakobranchus ocellatus.”  eLife 10:e60176.  2021.  The genome of a sea slug that keeps stolen chloroplasts, searched for algal photosynthesis genes.  None were found, though light still prolonged the starving slugs' lives.  Mirror:  PMC

1. 37a.

   Section Abstract:  “To settle the arguments, this study reported the genome of a kleptoplastic sea slug, Plakobranchus ocellatus, and found no evidence of photosynthetic genes encoded on the nucleus.” ↩

Martin, W. et al.  “Evolutionary analysis of Arabidopsis, cyanobacterial, and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus.”  Proceedings of the National Academy of Sciences 99(19):12246–12251.  2002.  A comparison of the 24,990 proteins of the plant Arabidopsis with three cyanobacterial and sixteen other prokaryotic genomes.  It extrapolates from the proteins conserved enough to compare to estimate how many plant nuclear genes came from the chloroplast's ancestor.  Mirror:  PMC

1. 38a.

   Section Abstract:  “Extrapolating from these conserved proteins to the whole genome, the data suggest that ≈4,500 of Arabidopsis protein-coding genes (≈18% of the total) were acquired from the cyanobacterial ancestor of plastids.” ↩

Thorsness, P. E., and T. D. Fox.  “Escape of DNA from mitochondria to the nucleus in Saccharomyces cerevisiae.”  Nature 346(6282):376–379.  1990.  A yeast genetics experiment that placed a nuclear marker gene on DNA kept inside mitochondria and counted how often it turned up working in the nucleus.  It gives the only per-cell, per-generation escape rate used here.

1. 39a.

   Section Abstract:  “Our results show that, in yeast, DNA escapes from mitochondria and appears in the nucleus at a surprisingly high frequency (approximately 2 x 10(-5) per cell per generation).” ↩

Huang, C. Y. et al.  “Direct measurement of the transfer rate of chloroplast DNA into the nucleus.”  Nature 422(6927):72–76.  2003.  A tobacco breeding experiment with a nuclear-type marker gene placed in the chloroplast genome.  About 250,000 seedlings fathered by such plants were screened, and 16 had inherited the marker in the nucleus.

1. 40a.

   Section Abstract:  “Thus, we provide a quantitative estimate of one transposition event in about 16,000 pollen grains for the frequency of transfer of cpDNA to the nucleus.” ↩

Stegemann, Sandra et al.  “High-frequency gene transfer from the chloroplast genome to the nucleus.”  Proceedings of the National Academy of Sciences 100(15):8828–8833.  2003.  A tobacco experiment that selected leaf cells in which a chloroplast marker gene had reached the nucleus, and estimated the frequency by dividing the number of leaf cells by the number of events.  The authors call it a lower bound.  Mirror:  PMC

1. 41a.

   Section Frequency of Gene Transfer:  “we obtain a frequency of approximately 1 of 5 million cells where transfer of the chloroplast nptII-containing fragment into the nuclear genome has occurred”. ↩

Wei, W. et al.  “Nuclear-embedded mitochondrial DNA sequences in 66,083 human genomes.”  Nature 611(7934):105–114.  2022.  A survey of mitochondrial DNA insertions in the nuclear genomes of 66,083 people in the UK 100,000 Genomes Project, including 8,201 complete mother-father-child trios, from which the rate of new insertions per generation was counted.  Mirror:  PMC

1. 42a.

   Section Results:  “Analysis of NUMT segregation in 8,201 complete mother–father–child trios revealed three private NUMTs from two families that were not seen in either parent”.  “The de novo NUMT frequency is likely to be an underestimate”.  The paper gives the rate as 2.44 × 10⁻⁴ per generation, with a 95% interval of 2.95 × 10⁻⁵ to 8.81 × 10⁻⁴. ↩ ↩

Stegemann, Sandra, and Ralph Bock.  “Experimental reconstruction of functional gene transfer from the tobacco plastid genome to the nucleus.”  The Plant Cell 18(11):2869–2878.  2006.  A tobacco experiment from Ralph Bock's laboratory that took plants already carrying a chloroplast gene in the nucleus and selected for the rare cells in which that gene became active.  It is the only measured rate of a moved organelle gene starting to work, and it shows how the gene was switched on.  Mirror:  PMC

1. 43a.

   Section Discussion:  “In our experimental system, nuclear activation of the transferred chloroplast gene occurred at a frequency of eight events in 5564 leaf explants subjected to selection”.  Section Abstract:  “Acquisition of gene function involves (1) transcriptional activation by capture of the promoter of an upstream nuclear gene and (2) utilization of AT-rich noncoding sequences downstream of the plastid gene as RNA cleavage and polyadenylation sites.”  Allowing for the number of cells in the leaf area, the authors put this at about 3 × 10⁻⁸ per cell. ↩

2. 43b.

   Section Discussion:  “the frequency of a chloroplast transgene escaping to the nucleus and becoming functional there would be in the range of”.  The range given is 10⁻¹⁴ to 10⁻¹⁵.  The authors add that the frequency may be higher in pollen. ↩

3. 43c.

   Section Discussion:  “It seems conceivable that the probability of functional gene transfer as determined in this work would be lower if acquisition of a transit peptide sequence for rerouting of the gene product into the chloroplast was a”. ↩

Fuentes, I. et al.  “Experimental reconstruction of the functional transfer of intron-containing plastid genes to the nucleus.”  Current Biology 22(9):763–771.  2012.  A follow-up tobacco experiment from Ralph Bock's laboratory showing that chloroplast genes with introns can become working nuclear genes, by using hidden splice sites inside the intron.

1. 44a.

   Section Abstract:  “intron possession is not an insurmountable obstacle to DNA-mediated endosymbiotic gene transfer”. ↩

Lynch, Michael et al.  “The divergence of mutation rates and spectra across the Tree of Life.”  EMBO Reports 24(10):e57561.  2023.  A review and compilation of measured mutation rates and diversity across 144 species, from which effective population sizes are calculated.  It is used here only for the range of population sizes of single-celled organisms.

1. 45a.

   Section Mutation‐rate variation in light of the drift‐barrier hypothesis:  “for any particular organism size, there is also an order‐of‐magnitude range of variation in”.  The same sentence puts the upper limit at about a billion even in the smallest microbes.  The median of about 27 million for the 16 single-celled eukaryotes is computed from the paper's Dataset EV1. ↩

The calculation treats each gene independently.  The per-gene rate u is the rate at which a working nuclear copy arises:  the low end and high end of Stegemann and Bock's combined figure, and the product of Huang's pollen escape rate with Stegemann and Bock's activation rate.  Each is multiplied by 0.027 for gaining a targeting tag, from Baker and Schatz's fraction of random DNA fragments that work as one.⁴⁷  A neutral copy fixes at rate u per generation (Kimura);  a favoured one at about 2Nsu, since its chance of fixing is about 2s.  The expected time for all n genes to finish is the nth harmonic number divided by the per-gene rate, about 7.5 divided by the rate for 1,000 genes.  Years assume one generation a day;  at the 14 to 25 days of the cultured Asgard archaeon, every figure is 14 to 25 times longer.  Two assumptions favour the theory:  the lab frequencies, measured per cell under antibiotic selection, are treated as per generation, and losing the old organelle copy is treated as free.  One cuts against it:  real genes may need no targeting tag when the protein works outside the organelle.  The script is transfer-arithmetic.ts. ↩

Baker, Alison, and Gottfried Schatz.  “Sequences from a prokaryotic genome or the mouse dihydrofolate reductase gene can restore the import of a truncated precursor protein into yeast mitochondria.”  Proceedings of the National Academy of Sciences 84(10):3117–3121.  1987.  A yeast experiment from Gottfried Schatz's laboratory, a founder of the study of mitochondrial protein import.  Random fragments of E. coli DNA were fused in front of a mitochondrial protein that had lost most of its address tag, and the fraction that restored import was counted by growth on a respiratory food source.  Mirror:  PMC

1. 47a.

   Page 3117:  “These sequences, which resemble authentic presequences in their overall amino acid composition and degree of hydrophobicity, are rather frequent; >2.7% of clones generated from E. coli DNA and >5% of clones from the dihydrofolate reductase gene were functional in our screening system.” ↩ ↩

Lucattini, R. et al.  “Bacterial proteins predisposed for targeting to mitochondria.”  Molecular Biology and Evolution 21(4):652–658.  2004.  A computer survey of the proteins of a living proteobacterium with two methods that predict mitochondrial targeting, checked on simulated genomes, plus an import test of one bacterial protein family on isolated mitochondria.  It asks whether the bacterium's proteins already carried usable address tags.

1. 48a.

   Section Abstract:  “According to current models for this evolution, two highly improbable events are required to occur simultaneously: creation of a protein translocation machinery to import proteins back into the endosymbiont and creation of targeting sequences on the protein substrates themselves. Using a combination of two independent prediction methods, validated through tests on simulated genomes, we show that at least 5% of proteins encoded by an extant proteobacterium are predisposed for targeting to mitochondria”. ↩

2. 48b.

   Section Abstract:  “This activity leaves the development of a primitive translocation channel in the outer membrane of the endosymbiont as a single hurdle to initiating the evolution of mitochondria.” ↩

Dolezal, P. et al.  “Evolution of the molecular machines for protein import into mitochondria.”  Science 313(5785):314–318.  2006.  A review of where each part of the mitochondrial protein-import machinery came from, by a group whose own laboratory work is on that machinery.  It separates the parts borrowed from the bacterium from the parts with no bacterial relative.

1. 49a.

   Section Abstract:  “Some of the preexisting protein translocation apparatus of the endosymbiont appears to have been commandeered, including molecular chaperones, the signal peptidase, and some components of the protein-targeting machinery. However, the protein translocases that drive protein import into mitochondria have no obvious counterparts in bacteria, making it likely that these machines were created de novo.” ↩

Liu, Z. et al.  “Tracing the evolution of the mitochondrial protein import machinery.”  Computational Biology and Chemistry 35(6):336–340.  2011.  A homology search for each of 35 subunits of the mitochondrial import machinery across prokaryotes and eukaryotes, used to date when each subunit appeared.

1. 50a.

   Section Abstract:  “The results indicated that 6 of the 35 subunits have homologs in prokaryote, suggesting that they were prokaryotic origin; the major subunit gains were occurred in the earliest stage of eukaryotic evolution”. ↩

Fukasawa, Yoshinori et al.  “Origin and Evolutionary Alteration of the Mitochondrial Import System in Eukaryotic Lineages.”  Molecular Biology and Evolution 34(7):1574–1586.  2017.  A survey of which import-machinery proteins are present in which eukaryote genomes, used to reconstruct the import machinery of the last common ancestor of eukaryotes.  Mirror:  PMC

1. 51a.

   Section Abstract:  “Our results indicate that presequence transport machinery was mostly established in the last eukaryotic common ancestor”.  Section Results:  “Tim44 has a bacterial origin”. ↩

Spang, A. et al.  “Complex archaea that bridge the gap between prokaryotes and eukaryotes.”  Nature 521(7551):173–179.  2015.  A genome assembled from environmental DNA in sea-floor sediment near the Loki's Castle vent field, naming the Lokiarchaeota.  Its phylogeny placed eukaryotes inside this archaeal group, and its gene list included many proteins previously known only from eukaryotes.  No cell was seen.  Mirror:  PMC

1. 52a.

   Section Presence of diverse and abundant ESPs:  “a significant part of the predicted proteome (175 proteins or 3.3%) was most similar to eukaryotic proteins”. ↩

Zaremba-Niedzwiedzka, K. et al.  “Asgard archaea illuminate the origin of eukaryotic cellular complexity.”  Nature 541(7637):353–358.  2017.  A survey of new environmental genomes that grouped the Lokiarchaeota with three related lineages as the Asgard archaea, and found among them more genes for eukaryote-type membrane-trafficking proteins.

1. 53a.

   Section Abstract:  “Asgard archaea affiliate with eukaryotes in phylogenomic analyses, and their genomes are enriched for proteins formerly considered specific to eukaryotes.”  “Our results expand the known repertoire of 'eukaryote-specific' proteins in Archaea, indicating that the archaeal host cell already contained many key components that govern eukaryotic cellular complexity.” ↩

Imachi, H. et al.  “Isolation of an archaeon at the prokaryote–eukaryote interface.”  Nature 577:519–525.  2020.  The first Asgard archaeon grown in the laboratory, after a methane-fed bioreactor run of more than 2,000 days and years of further enrichment.  The cells were imaged by electron microscopy and their metabolism worked out.  Mirror:  PMC

1. 54a.

   Section Abstract:  “Here we report the decade-long isolation of an Asgard archaeon related to Lokiarchaeota from deep marine sediment.” ↩

2. 54b.

   Section Abstract:  “Although eukaryote-like intracellular complexes have been proposed for Asgard archaea”.  “the isolate has no visible organelle-like structure.”  Section Results:  “The doubling time was estimated to be approximately 14–25 days.” ↩

3. 54c.

   Section Discussion:  “the model is not definitive and more studies on Asgard archaea and other deep-branching eukaryotes are required to contextualize the most probable steps that occurred.” ↩

Rodrigues-Oliveira, T. et al.  “Actin cytoskeleton and complex cell architecture in an Asgard archaeon.”  Nature 613:332–339.  2023.  Cryo-electron tomography of a second Asgard archaeon, Lokiarchaeum ossiferum, grown in an enriched culture.  It imaged an actin-like filament network through the cells and their protrusions, and looked for internal membranes.

1. 55a.

   Page 332:  “A long-range cytoskeleton extends throughout the cell bodies, protrusions and constrictions.”  Page 335:  “Except for two instances (Extended Data Fig. 7f), we did not observe internal membrane-bound compartments.” ↩

Vosseberg, J. et al.  “The emerging view on the origin and early evolution of eukaryotic cells.”  Nature 633:295–305.  2024.  A review of eukaryote origins by several of the researchers who described the Asgard archaea.  It sets out what the Asgard genomes and cultures show and what they do not.

1. 56a.

   Paragraph on the cultured Lokiarchaeia:  “First, Lokiarchaeia are only distantly related to the most likely sister clade of eukaryotes. Second, their genomes encode a distinct subset of ESPs, and they have diverged from the last common ancestor of all Asgard archaea for probably more than two billion years”. ↩ ↩

2. 56b.

   Opening paragraph:  “In the tree of life, the branch separating the first from the last common ancestor of all eukaryotes is long and lacks evolutionary intermediates.” ↩

Eme, L. et al.  “Inference and reconstruction of the heimdallarchaeial ancestry of eukaryotes.”  Nature 618:992–999.  2023.  A phylogenomic study with many new Asgard genomes that tests where eukaryotes branch, using models chosen to reduce known artefacts.  It also reconstructs the lifestyle of the Asgard ancestor.

1. 57a.

   Page 992:  “We find that eukaryotes are placed, with high confidence, as a well-nested clade within Asgard archaea and as a sister lineage to Hodarchaeales, a newly proposed order within Heimdallarchaeia.” ↩

Zhang, J. et al.  “Deep origin of eukaryotes outside Heimdallarchaeia within Asgardarchaeota.”  Nature 642(8069):990–998.  2025.  A phylogenomic study adding 223 Asgard genomes from Chinese coastal wetlands.  With recoded alignments and composition-aware models it places eukaryotes one level outside the Heimdallarchaeia, against Eme and colleagues.  Mirror:  PMC

1. 58a.

   Section Abstract:  “we robustly place eukaryotes within the Asgard archaea as a sister clade to Heimdallarchaeia.” ↩

Da Cunha, V. et al.  “Lokiarchaea are close relatives of Euryarchaeota, not bridging the gap between prokaryotes and eukaryotes.”  PLoS Genetics 13(6):e1006810.  2017.  A reanalysis of the 36-protein dataset that first placed eukaryotes inside the Lokiarchaeota, protein by protein.  It found that one protein carried the signal and argued that the placement is an artefact.  The original authors replied in 2018.  Mirror:  PMC

1. 59a.

   Section Abstract:  “Strikingly, removal of a single protein, the elongation factor EF2, is sufficient to break the Eukaryotes-Lokiarchaea affiliation.”  Section Concluding remarks:  “Our analyses demonstrate here that the specific affiliation between Eukarya and Lokiarchaeota previously described is most likely an artefact of genome reconstruction and phylogenetic analyses.” ↩

Koumandou, V. L. et al.  “Molecular paleontology and complexity in the last eukaryotic common ancestor.”  Critical Reviews in Biochemistry and Molecular Biology 48(4):373–396.  2013.  A review that reconstructs the cell systems of the last eukaryotic common ancestor one at a time, from cytoskeleton to nuclear transport, from their spread across living eukaryotes.  Mirror:  PMC

1. 60a.

   Section Reconstructing the LECA:  “LECA had a mitochondrion, meiotic machinery, a sophisticated and flexible metabolic capability, a fully differentiated endomembrane system, phagocytosis, actinomyosin and tubulin-based cytoskeletal systems together with a large complement of motor proteins, and finally a nucleus subcompartmentalised into hetero- and euchromatin, together with sophisticated nucleocytoplasmic transport.” ↩

Koonin, Eugene V.  “The origin and early evolution of eukaryotes in the light of phylogenomics.”  Genome Biology 11:209.  2010.  A review by a leading computational evolutionary biologist at the US National Center for Biotechnology Information of what genome comparisons say about the first eukaryotes, arguing for a highly complex common ancestor.  Mirror:  PMC

1. 61a.

   Section The last common ancestor of eukaryotes:  “A MP reconstruction based on phyletic patterns in clusters of orthologous genes of eukaryotes mapped 4,137 genes to LECA”. ↩

Richards, T. A. et al.  “Reconstructing the last common ancestor of all eukaryotes.”  PLoS Biology 22(11):e3002917.  2024.  A consensus statement by about thirty researchers on how to reconstruct the genes of the last eukaryotic common ancestor.  It treats that gene set as not yet settled.  Mirror:  PMC

1. 62a.

   Section Abstract:  “Once assembled, a robust LECA gene set will be a useful tool for evaluating alternative hypotheses about the origin of eukaryotes”. ↩

Embley, T. M., and W. Martin.  “Eukaryotic evolution, changes and challenges.”  Nature 440(7084):623–630.  2006.  A review by two leading students of early eukaryote evolution of how the search for primitive eukaryotes without mitochondria ended.

1. 63a.

   Section Abstract:  “The idea that some eukaryotes primitively lacked mitochondria and were true intermediates in the prokaryote-to-eukaryote transition was an exciting prospect. It spawned major advances in understanding anaerobic and parasitic eukaryotes and those with previously overlooked mitochondria. But the evolutionary gap between prokaryotes and eukaryotes is now deeper, and the nature of the host that acquired the mitochondrion more obscure, than ever before.” ↩

Karnkowska, A. et al.  “A Eukaryote without a Mitochondrial Organelle.”  Current Biology 26(10):1274–1284.  2016.  The genome of the gut flagellate Monocercomonoides, which lacks every hallmark mitochondrial protein and replaced the mitochondrial iron-sulfur pathway with a bacterial one taken by gene transfer.

1. 64a.

   Section Abstract:  “In the context of eukaryotic phylogeny, our data suggest that Monocercomonoides is not primitively amitochondrial but has lost the mitochondrion secondarily.” ↩

Pittis, A. A., and T. Gabaldón.  “Late acquisition of mitochondria by a host with chimaeric prokaryotic ancestry.”  Nature 531:101–104.  2016.  A phylogenomic study of 1,078 gene families present in the last eukaryotic common ancestor, comparing how far each sits from its closest prokaryotic relatives to infer the order in which they were acquired.  Mirror:  PMC

1. 65a.

   Section Abstract:  “In addition, the lack of evolutionary intermediates challenges the elucidation of the relative order of emergence of eukaryotic traits.” ↩

2. 65b.

   Section Abstract:  “Here we provide evidence for late mitochondrial endosymbiosis.” ↩

Vosseberg, J. et al.  “Timing the origin of eukaryotic cellular complexity with ancient duplications.”  Nature Ecology & Evolution 5(1):92–100.  2021.  A study that orders the gene duplications made before the last eukaryotic common ancestor by their branch lengths, to infer which cell systems expanded before and after the mitochondrion arrived.  Mirror:  PMC

1. 66a.

   Section Abstract:  “While evolutionary intermediates are lacking, gene duplications provide information on the order of events by which eukaryotes originated.”  “we infer that the host that engulfed the proto-mitochondrion had some eukaryote-like complexity, which drastically increased upon mitochondrial acquisition.” ↩ ↩

Martin, W. F. et al.  “Late Mitochondrial Origin Is an Artifact.”  Genome Biology and Evolution 9(2):373–379.  2017.  A reanalysis by William Martin's group of the data behind Pittis and Gabaldón's late-mitochondrion result.  Mirror:  PMC

1. 67a.

   Section Abstract:  “We show that the analyses underpinning a late mitochondrial origin suffer from multiple fatal flaws founded in inappropriate statistical methods and analyses, in addition to erroneous interpretations.” ↩

Martin, W., and E. V. Koonin.  “Introns and the origin of nucleus–cytosol compartmentalization.”  Nature 440(7080):41–45.  2006.  A hypothesis paper proposing that the nuclear envelope first arose to keep slow RNA splicing apart from fast protein synthesis, after introns spread from the newly captured mitochondrion.

1. 68a.

   Section Abstract:  “We propose that the nuclear envelope's incipient function was to allow mRNA splicing, which is slow, to go to completion so that translation, which is fast, would occur only on mRNA with intact reading frames.” ↩

Baum, D. A., and B. Baum.  “An inside-out origin for the eukaryotic cell.”  BMC Biology 12:76.  2014.  A hypothesis paper proposing that the eukaryotic cell grew outward from an archaeal cell, whose extruded blebs became the cytoplasm and whose original body became the nucleus.  It lists predictions by which the idea could be tested.  Mirror:  PMC

1. 69a.

   Section Overview of existing models of eukaryotic cell evolution:  “However, there is no evidence of endospore formation or other engulfment processes in Archaea, making this hypothesis improbable.” ↩

Lane, Nick, and William Martin.  “The energetics of genome complexity.”  Nature 467(7318):929–934.  2010.  An analysis by a biochemist and an evolutionary biologist arguing that the energy available per gene sets a ceiling on genome size, and that only cells with mitochondria could break it.

1. 70a.

   Section Abstract:  “The endosymbiosis that gave rise to mitochondria restructured the distribution of DNA in relation to bioenergetic membranes, permitting a remarkable 200,000-fold expansion in the number of genes expressed.”  “This vast leap in genomic capacity was strictly dependent on mitochondrial power, and prerequisite to eukaryote complexity: the key innovation en route to multicellular life.” ↩

Lynch, Michael, and Georgi K. Marinov.  “The bioenergetic costs of a gene.”  Proceedings of the National Academy of Sciences 112(51):15690–15695.  2015.  A calculation from published measurements of cell size, energy budgets and the DNA, RNA and protein made per gene, comparing what a gene costs a bacterium and a eukaryote.  Mirror:  PMC

1. 71a.

   Section Significance:  “These results eliminate the need to invoke an energetics barrier to genome complexity.” ↩

Lynch, Michael, and Georgi K. Marinov.  “Membranes, energetics, and evolution across the prokaryote-eukaryote divide.”  eLife 6:e20437.  2017.  A reanalysis of published figures on membrane area, energy production and cell size, arguing that eukaryotes are no more energy-efficient than prokaryotes.  The published reviews and the authors' replies are printed with it.  Mirror:  PMC

1. 72a.

   Section Abstract:  “However, contrary to this hypothesis, numerous lines of evidence suggest that eukaryotes are no more bioenergetically efficient than prokaryotes.”  Section Author response:  “The source of their repeated statements about a 200,000-fold expansion in genes and genome size eludes us”. ↩

Gerlitz, Marie et al.  “Elusive data underlying debate at the prokaryote-eukaryote divide.”  Biology Direct 13:21.  2018.  A check, by William Martin's group, of the literature values behind Lynch and Marinov's 2015 calculation.  It is a reply from one side of the dispute, not an independent audit.  Mirror:  PMC

1. 73a.

   Section Abstract:  “We report that for a sample of 80 numbers that were purportedly extracted from the literature and that underlie key inferences of the recent study, more than 50% of the values do not exist in the cited papers to which the numbers are attributed.”  “The published result cannot be independently reproduced.” ↩

Schavemaker, P. E., and S. A. Muñoz-Gómez.  “The role of mitochondrial energetics in the origin and diversification of eukaryotes.”  Nature Ecology & Evolution 6(9):1307–1317.  2022.  A model of the energy costs and limits of cells of different sizes and genome layouts, and of the cost and benefit to a host of carrying energy-producing symbionts.

1. 74a.

   Section Abstract:  “Thus, we argue that although mitochondria may not have been required by the first eukaryotes, eukaryote diversification was ultimately dependent on mitochondria.”  “a larger host cell may have derived a greater advantage (lower cost) from harbouring ATP-producing symbionts”. ↩

Rana, Fazale.  “Why Mitochondria Make My List of Best Biological Designs.”  Reasons to Believe.  May 1, 2019.  A blog essay by Fazale Rana of the old-earth ministry Reasons to Believe that builds a design argument on Nick Lane's account of why complex cells need mitochondria.

1. 75a.

   Paragraph on Lane's discovery:  “Through Lane's discovery, mitochondria reveal exquisite design and logical architecture and operations. Yet this is not necessarily what I (or many others) would have expected if mitochondria were the result of evolution.” ↩

Mehta, Angad P. et al.  “Engineering yeast endosymbionts as a step toward the evolution of mitochondria.”  Proceedings of the National Academy of Sciences 115(46):11796–11801.  2018.  A synthetic-biology experiment from Peter Schultz's laboratory at Scripps that engineered E. coli to live inside a respiration-deficient yeast, supplying it ATP in exchange for a vitamin, as a model of the first stage of mitochondrial evolution.  Mirror:  PMC

1. 76a.

   Section Abstract:  “Expression of several SNARE-like proteins in E. coli was also required, likely to block lysosomal degradation of intracellular bacteria. This chimeric system was stable for more than 40 doublings”. ↩

Cournoyer, J. et al.  “Engineering artificial photosynthetic life-forms through endosymbiosis.”  Nature Communications 13:2254.  2022.  A synthetic-biology experiment that placed engineered photosynthetic cyanobacteria inside respiration-deficient yeast and followed how long the combined cells kept growing on light.  Mirror:  PMC

1. 77a.

   Section Results:  “in our hands, we typically observed around 20 doublings on selection medium III”.  “As per our genomic DNA analysis of the chimeras, after more than 20 rounds of doublings, we observe that the chimeras lose their viability possibly due to loss of cyanobacteria”. ↩

Gao, Y. L. et al.  “Introducing carbon assimilation in yeasts using photosynthetic directed endosymbiosis.”  Nature Communications 15:5947.  2024.  A follow-up in which the cyanobacteria inside yeast were engineered to secrete sugar, so that the yeast could grow on carbon dioxide and light.  Mirror:  PMC

1. 78a.

   Section Abstract:  “Particularly, we engineer carbon assimilating and sugar-secreting photosynthetic cyanobacterial endosymbionts within the yeast cells”.  “chimeras that propagate under photosynthetic conditions”. ↩

Giger, G. H. et al.  “Inducing novel endosymbioses by implanting bacteria in fungi.”  Nature 635(8038):415–422.  2024.  An experiment from Julia Vorholt's laboratory at ETH Zurich that injected bacteria into fungal cells with a microscopic force-controlled needle, then selected spores carrying bacteria over ten rounds and measured how the partnership's cost fell.  Mirror:  PMC

1. 79a.

   Section Abstract:  “Whereas Escherichia coli implanted into the cytosol induced septum formation, effectively halting endosymbiogenesis, Mycetohabitans rhizoxinica was transmitted vertically to the progeny at a low frequency.”  “Continuous positive selection on endosymbiosis mitigated initial fitness constraints by several orders of magnitude upon adaptive evolution.”  Section Adaptive evolution of endosymbiosis:  “Whereas spores without bacteria had a germination success of 63%, the germination success of spores containing bacteria was reduced tenfold and was only 6.3%”. ↩ ↩

Agapakis, C. M. et al.  “Towards a Synthetic Chloroplast.”  PLoS ONE 6(4):e18877.  2011.  A synthetic-biology study that tested whether a photosynthetic cyanobacterium could live inside animal cells, by injection into zebrafish embryos and by engineering it to invade mammalian cells.  Mirror:  PMC

1. 80a.

   Section Abstract:  “In addition, when engineered with invasin from Yersinia pestis and listeriolysin O from Listeria monocytogenes, S. elongatus was able to invade cultured mammalian cells and divide inside macrophages.” ↩

Rasmussen, B. et al.  “Reassessing the first appearance of eukaryotes and cyanobacteria.”  Nature 455(7216):1101–1104.  2008.  Microscale carbon-isotope measurements on the Australian shales that had yielded the oldest claimed eukaryote and cyanobacteria biomarkers.  The isotopes showed the extracted molecules could not have come from the rock's own organic matter.

1. 81a.

   Section Abstract:  “These results are inconsistent with an indigenous origin for the biomarkers. Whatever their origin, the biomarkers must have entered the rock after peak metamorphism approximately 2.2 Gyr ago and thus do not provide evidence for the existence of eukaryotes and cyanobacteria in the Archaean eon.” ↩

2. 81b.

   Section Abstract:  “The oldest fossil evidence for eukaryotes and cyanobacteria therefore reverts to 1.78-1.68 Gyr ago and approximately 2.15 Gyr ago, respectively.” ↩

French, K. L. et al.  “Reappraisal of hydrocarbon biomarkers in Archean rocks.”  Proceedings of the National Academy of Sciences 112(19):5915–5920.  2015.  A multilaboratory study that drilled new cores in the same Australian rocks under hydrocarbon-clean conditions and compared them with older samples and blanks.

1. 82a.

   Section Abstract:  “Therefore, existing lipid biomarker evidence cannot be invoked to support the emergence of oxygenic photosynthesis and eukaryotes by ∼ 2.7 billion years ago.” ↩

Han, T.-M., and B. Runnegar.  “Megascopic eukaryotic algae from the 2.1-billion-year-old Negaunee iron-formation, Michigan.”  Science 257(5067):232–235.  1992.  A report of hundreds of coiled carbonaceous fossils from a Michigan iron formation, identified as the alga Grypania by their shape.

1. 83a.

   Section Abstract:  “As Grypania appears to have been a photosynthetic alga, this discovery places the origin of organelle-bearing eukaryotic cells prior to 2.1 billion years ago.” ↩

Miao, Lanyun et al.  “1.63-billion-year-old multicellular eukaryotes from the Chuanlinggou Formation in North China.”  Science Advances 10(4):eadk3208.  2024.  A description of cellularly preserved filament fossils from northern China, identified as eukaryotes by their size, structure and chemistry compared with living organisms.  Mirror:  PMC

1. 84a.

   Section Abstract:  “The fossils consist of large uniseriate, unbranched filaments with cell diameters up to 190 micrometers; spheroidal structures, possibly spores, occur within some cells.”  “In combination with spectroscopic characteristics, the large size and morphological complexity of these fossils support their interpretation as eukaryotes, likely photosynthetic, based on comparisons with extant organisms.” ↩

Butterfield, N. J.  “Bangiomorpha pubescens n. gen., n. sp.: implications for the evolution of sex, multicellularity, and the Mesoproterozoic/Neoproterozoic radiation of eukaryotes.”  Paleobiology 26(3):386–404.  2000.  The description of multicellular red-alga fossils from arctic Canada, identified by their pattern of cell division.

1. 85a.

   Section Abstract:  “Multicellular filaments from the ca. 1200-Ma Hunting Formation (Somerset Island, arctic Canada) are identified as bangiacean red algae on the basis of diagnostic cell-division patterns.”  “As the oldest taxonomically resolved eukaryote on record”. ↩

Gibson, Timothy M. et al.  “Precise age of Bangiomorpha pubescens dates the origin of eukaryotic photosynthesis.”  Geology 46(2):135–138.  2018.  A rhenium-osmium dating study of the sedimentary rocks above and below the Bangiomorpha fossils in the Bylot Supergroup of Baffin Island, which moved the alga's first appearance about 150 million years younger on the conventional timescale.  With the new age as a calibration, the authors' molecular clocks put the plastid endosymbiosis, the origin of photosynthesis in eukaryotes, at about 1.25 billion years. ↩

1. 86a.

   Section Abstract:  “revise its first appearance to 1.047 +0.013/–0.017 Ga. This date is 150 m.y. younger than commonly held interpretations”. ↩ ↩

Brocks, J. J. et al.  “Lost world of complex life and the late rise of the eukaryotic crown.”  Nature 618(7966):767–773.  2023.  A study of molecular fossils in mid-Proterozoic rocks that found early-stage sterols, "protosteroids," overlooked before, and used them to date when modern eukaryotes became common.

1. 87a.

   Section Abstract:  “This scarcity of eukaryotic remains is difficult to reconcile with molecular clocks that suggest that the last eukaryotic common ancestor (LECA) had already emerged between around 1,200 and more than 1,800 million years ago.”  “The protosteroids reveal an ecologically prominent 'protosterol biota' that was widespread and abundant in aquatic environments from at least 1,640 to around 800 million years ago and that probably comprised ancient protosterol-producing bacteria and deep-branching stem-group eukaryotes. Modern eukaryotes started to appear in the Tonian period (1,000 to 720 million years ago)”. ↩

Brocks, J. J. et al.  “The rise of algae in Cryogenian oceans and the emergence of animals.”  Nature 548(7669):578–581.  2017.  A record of eukaryote steroid molecules in rocks from before and after the Cryogenian glaciations, tracing when algae replaced bacteria as the main producers in the sea.

1. 88a.

   Section Abstract:  “Here we present a molecular fossil record of eukaryotic steroids demonstrating that bacteria were the only notable primary producers in the oceans before the Cryogenian period (720-635 million years ago).” ↩

Björkholm, Patrik et al.  “Mitochondrial genomes are retained by selective constraints on protein targeting.”  Proceedings of the National Academy of Sciences 112(33):10154–10161.  2015.  A laboratory study that predicted hydrophobic membrane proteins made from mitochondrial DNA would be sent to the endoplasmic reticulum if their genes were moved to the nucleus.  The authors expressed three of them in HeLa cells, found they went to the endoplasmic reticulum, and compared protein domains across bacteria and eukaryotes.  It is cited here only for its count of mitochondrial proteins made from nuclear genes.

1. 89a.

   Page 10154:  “In striking contrast to the few proteins encoded by the mitochondrial genome, it is estimated that more than 1,000 mitochondrial proteins are encoded by the nuclear genome (18).” ↩

Mitchell, Elizabeth.  “Winding Back Life's Story: Evolution of Mitochondria.”  Answers in Genesis.  Apr 7, 2016.  A young-earth news commentary on the Answers in Genesis site about Pittis and Gabaldón's 2016 paper on the late arrival of mitochondria.  It accepts "some superficial similarities" between mitochondria and bacteria and rejects the evolutionary account.

1. 90a.

   Paragraph comparing nuclear and mitochondrial DNA:  “Nuclear DNA is in the form of double helical strands, but mitochondrial DNA is not.” ↩