Major Evolutionary Transitions

Ratcliff, William C. et al.  “Experimental evolution of multicellularity.”  Proceedings of the National Academy of Sciences 109(5):1595–1600.  2012.  The first snowflake-yeast experiment, from Michael Travisano's laboratory at the University of Minnesota.  Ten, then five more, populations of a single-celled brewer's yeast were selected daily for fast settling for 60 days, and all became clusters of attached cells.  The paper measures the clusters' fitness, life cycle and rate of programmed cell death, but does not identify the genes involved.  Mirror:  PMC

1. 1a.

   Section Discussion:  “Although known transitions to complex multicellularity, with clearly differentiated cell types, occurred over millions of years …, we have shown that the first crucial steps in the transition from unicellularity to multicellularity can evolve remarkably quickly under appropriate selective conditions.” ↩

2. 1b.

   Section Discussion:  “Multicelled snowflake-phenotype yeast evolved in all 15 replicate populations, in two separate experiments, within 60 d of settling selection.” ↩ ↩

3. 1c.

   Section Discussion:  “This is functionally analogous to germ-soma differentiation”. ↩

4. 1d.

   Section Introduction, last paragraph:  “Ten replicate populations of initially isogenic S. cerevisiae were grown in nutrient-rich liquid medium with shaking to stationary phase (∼10^9 cells/replicate population) before subculturing and daily transfer to fresh medium.” ↩

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 cellular filaments with possible spores from shales dated about 1,635 million years, identified as eukaryotes from their size, shape and chemistry.  The introduction summarises the fossil record of early multicellularity.  Mirror:  PMC

1. 2a.

   Section Discussion:  “While simple multicellular eukaryotes evolved early, complex multicellular organisms did not appear for another billion years”.  “Therefore, while simple multicellularity may be necessary for the evolution of complex multicellular organisms, it is not sufficient.” ↩ ↩

2. 2b.

   Section Abstract:  “The occurrence of multicellular eukaryotes in Paleoproterozoic rocks not much younger than those containing the oldest unambiguous evidence of eukaryotes as a whole supports the hypothesis that simple multicellularity arose early in eukaryotic history, as much as a billion years before complex multicellular organisms diversified in the oceans.” ↩

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. 3a.

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

Swindell, Rick.  “Shining light on the evolution of photosynthesis.”  Journal of Creation 17(3):74–84.  Dec 2003.  The fullest young-earth treatment of photosynthesis found, by a plant geneticist, in Creation Ministries International's journal and reprinted by Answers in Genesis.  It walks through chlorophyll synthesis, the two photosystems, the Calvin cycle and oxygen protection, and argues that the whole system had to arise at once and so was designed.  It does not discuss how the manganese cluster could have arisen.  Mirror:  Answersingenesis.org

1. 4a.

   Page 76:  “PSII produces extremely strong oxidizing agents that can pull electrons out of water, but it is not capable of reducing NADP+ to NADPH. PSI produces extremely strong reducing agents that ultimately do the job of reducing ferredoxin and NADP+. Neither system does anything meaningful apart from the other, which is to say, nothing works unless everything works.” ↩ ↩ ↩

2. 4b.

   Page 78:  “Evolutionists maintain that ground-state oxygen (3O2, a triplet state biradical) was not around when photosynthesis evolved. There is, however, considerable evidence that there has never been a time in Earth's history when there was not significant free oxygen in the atmosphere”.  Swindell's footnotes for this cite Dimroth and Kimberley 1976, three creationist and design books, and Blankenship's 2002 textbook. ↩

Gibson, Amanda K. et al.  “The two-fold cost of sex: Experimental evidence from a natural system.”  Evolution Letters 1(1):6–15.  2017.  A field experiment, the first direct measurement of the cost of sex in a natural population.  Snails from Lake Alexandrina, New Zealand, with sexual and clonal females mixed, were raised for one generation in six outdoor tanks a year for four years, and the change in the clonal share was fitted to Maynard Smith's model.  The estimated cost was about two.  Mirror:  PMC

1. 5a.

   Page 12:  “The likelihood of model 3 was maximized at a cost of sex that slightly exceeds two (c = 2.14, 95% CI [1.81, 2.55])”. ↩ ↩

Cardona, Tanai et al.  “Early Archean origin of Photosystem II.”  Geobiology 17(2):127–150.  2019.  A molecular-clock study of the core proteins of all type II reaction centres, oxygenic and not, that puts the first water-splitting photosystem far earlier than the cyanobacteria.  The authors say the dates are approximate and that the early rates they require are hard to square with the proteins' structure.  Mirror:  PMC

1. 6a.

   Section Introduction:  “There is no doubt that D1, D2, L, and M share a common origin”.  Section 2.1 Change in sequence identity as a function of time:  “Any standard D1 or D2 shares on average 17% sequence identity with any L or M. The level of sequence identity falls well below 10% if any Type II reaction center protein is compared with any Type I reaction center protein”.  D1 and D2 are the core of Photosystem II;  L and M are the core of the purple bacteria's reaction centre. ↩ ↩

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. 7a.

   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.” ↩ ↩ ↩

Prochnik, Simon E. et al.  “Genomic analysis of organismal complexity in the multicellular green alga Volvox carteri.”  Science 329(5988):223–226.  2010.  The Volvox carteri genome, compared gene by gene with that of its single-celled relative Chlamydomonas reinhardtii by a large consortium.  The two have similar numbers of genes and almost the same developmental and regulatory proteins;  the differences lie mainly in enlarged families of matrix proteins and in a few genes such as regA.  The supporting text reviews which Volvox developmental genes can be replaced by their Chlamydomonas versions.  Mirror:  PMC

1. 8a.

   Section Abstract:  “Despite fundamental differences in organismal complexity and life history, the two species have similar protein-coding potentials, and few species-specific protein-coding gene predictions.” ↩ ↩

Carroll, Sean B.  Endless Forms Most Beautiful: The New Science of Evo Devo and the Making of the Animal Kingdom.  W. W. Norton, New York.  2005.  A popular book by a leading developmental geneticist on how a shared kit of developmental genes builds very different animals, and what that means for evolution.

1. 9a.

   Pages 284–285:  “Furthermore, once the structures of DNA and proteins were understood, the prevailing view of the architects and adherents of Modern Synthesis was that the process of random mutation and selection would so alter DNA and protein sequences that only closely related species would bear homologous genes.”  Page 285:  “The first and still perhaps the most stunning discovery of Evo Devo is the ancient origin of the genes for building all sorts of animals (chapters 3 and 6). The fact that such different forms of animals are shaped by very similar sets of tool kit proteins was entirely unanticipated.” ↩

Blankenship, Robert E.  Molecular Mechanisms of Photosynthesis.  Blackwell Science, Oxford.  2002.  The standard graduate textbook on photosynthesis by a Washington University biochemist who has written widely on its evolution.  Swindell cites it for his oxygen argument.

1. 10a.

   Page 225:  “Geological evidence strongly suggests that the atmosphere contained significant quantities of O2 by about 2.2 billion years ago (Des Marais et al., 1992; Rye and Holland, 1998: Canfield et al., 2000). This represents the latest date for the advent of oxygenic photosynthesis. However, considerable evidence suggests that O2 evolution may have started significantly earlier, but that it took some time for free O2 to accumulate to significant levels in the atmosphere.” ↩ ↩ ↩

2. 10b.

   Page 134:  “Green sulfur bacteria have a single photosystem similar to photosystem 1, such that it can directly reduce ferredoxin and then NAD+ without the need for energy-dependent reverse electron flow.” ↩

3. 10c.

   Page 143:  “Ferredoxin-catalyzed cyclic electron flow around photosystem 1 was first observed by Arnon and coworkers in 1954 (Arnon, 1984). It is readily activated if electron flow from photosystem 2 is cut off, for example with the inhibitor DCMU”.  On p. 144 Blankenship adds that in normal leaves cyclic flow is "at most a few percent under optimum conditions, but may increase dramatically under stress conditions." ↩

Butterfield, Nicholas 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 a fossil red alga from arctic Canada, identified by its pattern of cell division, and read as the oldest sexually reproducing organism known from fossils.

1. 11a.

   Section abstract:  “Differential spore/gamete formation shows Bangiomorpha pubescens to have been sexually reproducing, the oldest reported occurrence in the fossil record.” ↩

2. 11b.

   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 rocks above and below the Bangiomorpha fossils on Baffin Island, revising their age downward by 150 million years on the conventional timescale.

1. 12a.

   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”. ↩ ↩

Allen, James P. et al.  “Light-driven oxygen production from superoxide by Mn-binding bacterial reaction centers.”  Proceedings of the National Academy of Sciences 109(7):2314–2318.  2012.  A laboratory study from the Arizona State group that has spent two decades redesigning the reaction centre of the purple bacterium Rhodobacter sphaeroides to resemble Photosystem II.  Their mutant centres, given a stronger oxidant and a manganese site, turn superoxide into oxygen in light.  None of them oxidises water.  Mirror:  PMC

1. 13a.

   Section Materials and Methods:  “The Mn-binding mutant M8 has three changes near P to increase the oxidation/reduction potential, Leu L131 to His, Leu M160 to His, and Phe M197 to His, and three changes that create the Mn-binding site, Arg M164 to Tyr, Met M168 to Glu, and Gly M188 to Asp.”  P is the pair of bacteriochlorophylls that gives up an electron when light strikes.  In the published sequence of the Rhodobacter sphaeroides M protein, residue 188 is an asparagine and residue 288 is a glycine, so "M188" may be a misprint for M288. ↩ ↩

2. 13b.

   Section Abstract:  “Unlike wild-type reaction centers, the highly oxidizing reaction centers are not stable in the light unless they have a bound Mn.” ↩

Martin, William F. et al.  “A physiological perspective on the origin and evolution of photosynthesis.”  FEMS Microbiology Reviews 42(2):205–231.  2018.  A long review by an evolutionary biochemist and two photosynthesis researchers that builds a step-by-step scenario from the first chlorophyll to two photosystems in series.  It is unusually plain about which steps rest on no observed organism.  Mirror:  PMC

1. 14a.

   Section Origin of chlorophototrophy in cyanobacteria:  “there are no bacterial lineages known that branch immediately ancestral to cyanobacteria and that possess only one RC or some other 'primitive' form of photosynthesis.”  RC means reaction centre. ↩ ↩

2. 14b.

   Section Introduction:  “There is, however, no consensus concerning the physiological processes that mediated either transition.”  The two transitions are the origin of chlorophyll-based photosynthesis and the origin of oxygenic photosynthesis. ↩

Cardona, Tanai et al.  “Origin and evolution of water oxidation before the last common ancestor of the Cyanobacteria.”  Molecular Biology and Evolution 32(5):1310–1328.  2015.  A comparison of D1 protein sequences across cyanobacteria and the related bacterial reaction centres, which sorts D1 into four groups and argues that water splitting arose before the last common ancestor of living cyanobacteria.  It gives no dates.  Mirror:  PMC

1. 15a.

   Section Introduction:  “it is still unclear how an ancestral bacterium evolved the capacity to split water”. ↩ ↩

The calculation is the photosynthesis article's own.  It prices the six changes of the designed manganese centre, which need at least 11 DNA base changes, 5 of them for the manganese site.¹³  It uses a cyanobacterial mutation rate of 3.5 × 10⁻¹⁰ per base per generation, one generation a day, and an advantage of 1% per step where selection is allowed.  If each change helped, the 5 site changes spread in about 80 to 400 years and all 11 in about 170 to 870 years.  If each was neutral, about 54 and 71 million years.  If all had to arrive together, by Behe and Snoke's equation with their own ratio of 1,000 useless mutations to each useful one, the 5 site changes take about 29,000 years in a population the size of today's ocean cyanobacteria, about 3 × 10²⁷ cells, and about 1.2 × 10¹⁷ years at the effective population size of 1.68 × 10⁷;  all 11 take about 8.6 × 10¹⁶ years even at the census size.  The truth for long periods lies between the census and effective-size figures. ↩ ↩ ↩ ↩

Oliver, Thomas et al.  “The Evolution and Evolvability of Photosystem II.”  Annual Review of Plant Biology 74:225–257.  2023.  A review by the Imperial College group that argues Photosystem II and water splitting are very old, older than the cyanobacteria.  It is the best statement of the early-origin view, and it is candid about how weak the dating is.

1. 17a.

   Section 3. Origin of Photosystem II and water oxidation:  “molecular clock studies have provided a range of dates spanning two billion years between 3.5 and 1.5 Ga”. ↩ ↩

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. 18a.

   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.” ↩

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. 19a.

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

2. 19b.

   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.” ↩ ↩

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. 20a.

   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?” ↩

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. 21a.

   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. 22a.

   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. 22b.

   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. 22c.

   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”. ↩

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. 23a.

   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.” ↩

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. 24a.

   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.” ↩

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. 25a.

   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.” ↩

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. 26a.

   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.” ↩

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. 27a.

   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. 28a.

   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.” ↩

The calculation is the endosymbiosis article's own, and treats each gene on its own.  A working nuclear copy of an organelle gene arises at rates measured in tobacco and yeast, multiplied by 0.027 for the chance that a random stretch of DNA works as a mitochondrial address tag.³⁰  A neutral copy fixes at that rate per generation, and a favoured one about 2Ns times faster, where N is the population size and s the advantage.  At one generation a day, drift alone takes about 54 billion years for one addressed gene at the best measured rate.  With s = 0.01, a thousand genes relocate in about 200,000 years at a population of 10⁸ and the high rate, and about 3.8 billion years at 10⁷ and the low rate.  The population sizes follow Michael Lynch's survey of single-celled eukaryotes, whose median is about 27 million.⁸³  The laboratory frequencies were measured per cell under antibiotic selection and are treated as per generation, which favours the theory. ↩ ↩ ↩ ↩

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. 30a.

   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.” ↩ ↩

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. 31a.

   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”. ↩

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. 32a.

   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.” ↩

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. 33a.

   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.” ↩

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. 34a.

   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.” ↩ ↩

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. 35a.

   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.” ↩ ↩

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. 36a.

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

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. 37a.

   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.” ↩

Bell, Graham.  The Masterpiece of Nature: The Evolution and Genetics of Sexuality.  University of California Press.  1982.  A long monograph by a McGill evolutionary biologist that sets out the hypotheses for the maintenance of sex and tests them against the distribution of sex and parthenogenesis across living things.  Its opening sentence is the most quoted line in creationist writing on sex.

1. 38a.

   Page 19:  “Sex is the queen of problems in evolutionary biology. Perhaps no other natural phenomenon has aroused so much interest; certainly none has sowed as much confusion.” ↩

2. 38b.

   Page 19:  “The largest and least ignorable and most obdurate of these questions is, why sex? Or, to put this more technically, what is the functional significance of sexuality, which leads to its maintenance under natural selection in biological populations?” ↩ ↩

Jokela, Jukka et al.  “The maintenance of sex, clonal dynamics, and host-parasite coevolution in a mixed population of sexual and asexual snails.”  The American Naturalist 174(S1):S43–S53.  2009.  A long-term field study of one mixed population of sexual and clonal mud snails, following which clones were common and how infected they were over 7 to 10 years.  It is the first direct test of the Red Queen's prediction in competing sexual and asexual lineages in nature.

1. 39a.

   Section abstract:  “We found that, within 7-10 years, the most common clones were almost completely replaced by initially rare clones in two different habitats, while sexuals persisted throughout the study period.” ↩

Morran, Levi T. et al.  “Running with the Red Queen: host-parasite coevolution selects for biparental sex.”  Science 333(6039):216–218.  2011.  A laboratory evolution experiment with the nematode Caenorhabditis elegans, comparing populations forced to self, forced to outcross, or free to do either, under no pathogen, a fixed pathogen, or a coevolving pathogen, for about 30 generations.  Page numbers are those of the author manuscript.  Mirror:  PMC

1. 40a.

   Section author manuscript p. 3:  “In the coevolution treatment, all of the obligately selfing populations became extinct within twenty generations (Fig S1).”  “Over the first eight generations of the experiment, outcrossing rates increased from 20% to over 70% in both the evolution and coevolution treatments”.  “all of the obligately outcrossing and wildtype populations persisted throughout the experiment in all three treatment types (Fig S1).” ↩

Goddard, Matthew R. et al.  “Sex increases the efficacy of natural selection in experimental yeast populations.”  Nature 434:636–640.  2005.  A laboratory evolution experiment with eight sexual and eight asexual yeast populations, the asexual strain lacking two meiosis genes, kept in a benign and a harsh environment with periodic sporulation.  Sex sped adaptation in the harsh environment only.

1. 41a.

   Page 638:  “In the asexual lines, relative fitness increased from 0 to 0.59, whereas for the sexual lines the increase was from 0 to 0.68. These figures are equivalent to increases in population growth rate of 80% and 94%, respectively.”  “The difference in fitness between the sexual and asexual populations after 100 generations, and indeed throughout most of the experiment, is about 0.1.”  “Thus in the 25 mitotic generations between episodes of sex in our experiments, a sexual individual could expect to leave about 12-fold”.  Relative fitness is defined on the same page as the natural logarithm of growth rate relative to the ancestor.  The printed sentence ends with the expression e^(0.1×25), which the scan renders poorly. ↩ ↩

Otto, Sarah P., and Scott L. Nuismer.  “Species Interactions and the Evolution of Sex.”  Science 304(5673):1018–1020.  2004.  A population-genetic model of the Red Queen across 13,608 combinations of selection strength, recombination, mortality and seven kinds of species interaction.  It includes no cost of sex, and the authors note that adding one would only shrink the conditions favouring sex.

1. 42a.

   Page 1018:  “Our results show that species interactions typically select against sex. We conclude that, although the Red Queen favors sex under certain circumstances, it alone does not account for the ubiquity of sex.” ↩

The bound is the sex article's own.  A clone's mean fitness at equilibrium is e^(−U) times that of its best class, where U is the number of new harmful mutations per genome per generation.⁸⁴  Neither a clone's best class nor a sexual population can be fitter than a mutation-free genome, so sex can raise mean fitness at most e^U-fold.  Paying a cost of two needs e^U of at least 2, or U of at least ln 2 = 0.693.  For yeast, Goddard and colleagues' advantage of about 0.1 in log growth rate per generation is a factor of 1.105.⁴¹  Against a twofold cost paid every generation it covers ln 1.105 ÷ ln 2, about 14%.  Paid once in 25 generations, as in the experiment, the gain is about 12-fold and pays the cost about six times over.  The yeast have no males, so this assumes the advantage would carry over to an animal, which is generous to the hypothesis. ↩ ↩ ↩

Keightley, Peter D., and Adam Eyre-Walker.  “Deleterious Mutations and the Evolution of Sex.”  Science 290(5490):331–333.  2000.  A comparative genomic test of Kondrashov's hypothesis by two population geneticists.  It estimates the rate of harmful mutation in protein-coding genes for nine pairs of animal species from their divergence, and finds the rate per generation rises with generation time, falling far below one in short-lived animals.  Its gene counts predate the human genome sequence, which roughly quarters the vertebrate figures.

1. 44a.

   Paragraph 5:  “However, many obligate sexuals have generation times on the order of 1 month or less [e.g., many insects ( 16 ), copepods ( 17 ), and nematodes ( 18 )], implying deleterious amino acid mutation rates of the order of 0.05, some 20 times lower than the MD hypothesis requires.”  Paragraph last:  “However, based on our deleterious mutation rate estimates, the hypothesis is not supported as a general mechanism.”  MD is the mutational deterministic hypothesis.  The 0.8 for the whole fruit-fly genome is from the paragraph on noncoding DNA. ↩ ↩

Malik, Shehre-Banoo et al.  “An Expanded Inventory of Conserved Meiotic Genes Provides Evidence for Sex in Trichomonas vaginalis.”  PLoS ONE 3(8):e2879.  2008.  A search of more than 30 eukaryote genomes for 29 meiosis genes.  Trichomonas had 27 and Giardia 21, though neither has been seen in meiosis.  The authors infer the genes were in the common ancestor of all eukaryotes, and note that missing genes do not prove asexuality.

1. 45a.

   Section Conclusions:  “These 27 meiotic genes must have been present in the common ancestor of Trichomonas and Giardia, and given the highly diverged positions of these lineages among eukaryotes [12–16], each of the genes also must have been present in the common ancestor of all eukaryotes.” ↩

Lenormand, Thomas et al.  “Evolutionary mysteries in meiosis.”  Philosophical Transactions of the Royal Society B 371:20160001.  2016.  A review of the features of meiosis whose origin and function are unexplained, introducing a themed issue.  It sets out the rival hypotheses for each step and the objections to each.

1. 46a.

   Page 1:  “The origin of meiosis through gradual steps is among the most intriguing evolutionary enigmas”.  Page 2:  “This scenario presupposes that DNA maintenance is inefficient in the absence of meiosis; however, prokaryotes (including archaea) have efficient repair mechanisms that involve recombination, but not meiosis”. ↩ ↩

Wilkins, Adam S., and Robin Holliday.  “The Evolution of Meiosis From Mitosis.”  Genetics 181(1):3–12.  2009.  A Perspectives essay by a developmental geneticist and the discoverer of the Holliday junction, proposing that meiosis evolved from ordinary cell division with chromosome pairing as its first new step.  It presents no data and says the hypothesis cannot be tested directly.  Mirror:  PMC

1. 47a.

   Page 3:  “origins of meiosis in early eukaryotic history have never been satisfactorily explained.”  Page 9:  “There is, of course, no direct way to test the basic hypothesis presented here since the cells in which meiosis first originated existed well over 1 billion years ago”. ↩

Brown, Walter T.  In the Beginning: Compelling Evidence for Creation and the Flood.  Center for Scientific Creation.  8th edition.  2008.  A young-earth book by a retired engineer and creationist author, arranged as short numbered arguments with endnotes.  Section 37, "Sexual Reproduction," argues that the two sexes could not have evolved step by step, and quotes several scientists on the difficulty of explaining sex.

1. 48a.

   Page 20:  “The amazingly complex, radically different, yet complementary reproductive systems of the male and female must have completely and independently evolved at each stage about the same time and place. Just a slight incompleteness in only one of the two at any stage would make both reproductive systems useless, and the organism would become extinct.”  Item a of section 37. ↩ ↩

Geng, Sa et al.  “Evolution of Sexes from an Ancestral Mating-Type Specification Pathway.”  PLoS Biology 12(7):e1001904.  2014.  A genetic experiment in the alga Volvox carteri, which has eggs and sperm.  The mating-type gene MID, which in its single-celled relative Chlamydomonas sets the minus mating type, was added to females and knocked down in males to see whether it now controls sex.

1. 49a.

   Section abstract:  “Transgenic female V. carteri expressing male MID produced functional sperm packets during sexual development.”  “produced functional eggs, or self-fertile hermaphrodites”. ↩

Behe, Michael J.  “More Darwinian Degradation.”  Michael J. Behe's Blog, Uncommon Descent.  Jan 24, 2012.  A two-paragraph blog post by the Lehigh University biochemist and intelligent-design author, written on the first snowflake-yeast paper before its genetics were known.  It summarises the experiment and predicts that the genes behind it will prove to be broken.  Mirror:  Archive.org

1. 50a.

   Second paragraph:  “In fact, the results can be regarded as the loss of two pre-existing abilities: 1) the loss of the ability to separate from the mother cell during cell division; and 2) the loss of control of apoptosis. The authors did not analyze the genetic changes that occurred in the cells, but I strongly suspect that if and when they do, they’ll discover that functioning genes or regulatory regions were broken or degraded.” ↩

Ratcliff, William C. et al.  “Origins of multicellular evolvability in snowflake yeast.”  Nature Communications 6:6102.  2015.  The follow-up that found the genetic cause of the 2012 snowflake yeast.  The authors measured gene expression in an early snowflake strain, sequenced the gene ACE2 in one isolate from each of the ten populations, deleted it from the ancestor and restored it in an evolved strain, and modelled how the cluster shape affects heritability.  Mirror:  PMC

1. 51a.

   Page 3:  “Further, we found that complementation of an experimentally evolved snowflake yeast (population 1, codon 645val * asp) with a single copy of functional ACE2 linked to KANMX4 resulted in reversion to unicellularity (Fig. 1d).”  The same paragraph reports that a knockout of ACE2 in the single-celled ancestor produced the snowflake form. ↩

Answers in Genesis.  “Multicellular Life Grows In Test Tube.”  Answers in Genesis, News to Note.  Jul 2, 2011.  An unsigned young-earth news item on the first conference report of the snowflake-yeast experiment, arguing that selection only uncovered abilities the yeast already had.

1. 52a.

   Near the end:  “Thus, genetic information to become multicellular did not evolve in Ratcliff’s laboratory; the information was in the genome all along.” ↩

Mitchell, Elizabeth.  “Yeast Begat Yeast.”  Answers in Genesis, News to Note.  Jan 21, 2012.  A young-earth review of the published 2012 snowflake-yeast paper writing for Answers in Genesis.  It argues that the yeast already had the snowflake form and evolved nothing new.

1. 53a.

   Second section:  “They demonstrated the yeast had this characteristic behavior before beginning the experiment.” ↩ ↩

Bozdag, G. Ozan et al.  “De novo evolution of macroscopic multicellularity.”  Nature 617:747–754.  2023.  The first report of the multicellularity long-term evolution experiment, from Ratcliff's laboratory at the Georgia Institute of Technology.  Fifteen snowflake-yeast populations, already lacking ACE2, were selected daily for size for 600 days, under three oxygen regimes.  The oxygen-free populations became large enough to see, through longer cells that tangle; the paper sequenced one strain from each population.  Mirror:  PMC

1. 54a.

   Section Results:  “Yeast in this treatment increased their mean cluster radius from 16 μm to 434 μm, a ~2·10^4-fold increase in volume (Fig. 1e). This corresponds to an estimated increase from ~100 cells per cluster to ~450,000”.  The abstract gives the same ~2·10^4 figure;  the introduction says ~2·10^5, which the radii do not support. ↩ ↩

2. 54b.

   Section Introduction:  “a model of undifferentiated multicellularity”.  “creating opportunities for the evolution of cellular differentiation”. ↩

Umen, James G.  “Green algae and the origins of multicellularity in the plant kingdom.”  Cold Spring Harbor Perspectives in Biology 6(11):a016170.  2014.  A review of multicellularity in the green algae by a plant biologist who works on Chlamydomonas and Volvox.  It covers the volvocine series, Kirk's twelve steps and the genes known to act in them, and questions the common claim that simple multicellularity is the easy step.  Mirror:  PMC

1. 55a.

   Section Multicellular Innovations in the Volvocine Lineage, steps 11 and 12:  “As described above, the genus Volvox is polyphyletic with germ-soma differentiation established by different mechanisms in separate groups”. ↩

Gauger, Ann.  “A Simple Transition to Multicellularity -- Not!”  Evolution News, Discovery Institute.  May 7, 2015.  An intelligent-design blog post by a biologist and senior fellow of the Discovery Institute's Center for Science and Culture, listing what the evolutionary story of Volvox would need and arguing that the details are missing.

1. 56a.

   “A theory has been proposed involving 12 steps for how this change from single-celled to multicellular Volvox might have happened. Missing from this story, though, are the details necessary for this 12-step progression to occur.” ↩

Doyle, Shaun.  “Evolution of multicellularity: what is required?”  Journal of Creation 23(1):5–7.  2009 (edition of 2010).  A young-earth article from Creation Ministries International, written before the yeast experiments, that lists what the author takes true multicellularity to require and argues that colonies such as Volvox fall short.

1. 57a.

   Section Requirements for multicellular evolution, Genetic sameness:  “Evolution requires more than a mere augmentation of an existing system for co-ordinated multicellularity to evolve; it requires the ex nihilo creation of an entirely new system of organisation to co-ordinate cells appropriately to form a multicellular individual.” ↩

The arithmetic is the multicellularity article's own.  Yeast makes about 0.0042 new mutations per cell division, from a rate of 1.67 × 10⁻¹⁰ per base over a genome of 12.5 million bases in two copies.  A population regrowing to about 10⁹ cells a day¹ makes about 6 × 10¹⁰ divisions in 60 days, enough to produce each of the roughly 3.7 × 10⁷ possible single-letter changes about seven times.  On Herron and colleagues' clock, about 107 to 146 million years separate the first volvocine colonies from the first sterile body cells.⁸⁵  How often wild algae divide is not measured.  One division a week to one a day, an assumed range, gives about 6 billion to 50 billion generations, about one million to ten million times the roughly 5,000 generations of the longest yeast experiment.⁸⁶  A young-earth window of 4,500 to 6,000 years gives about 230,000 to 2.2 million generations, still 50 to 400 times the longest experiment. ↩ ↩ ↩ ↩

King, Nicole et al.  “The genome of the choanoflagellate Monosiga brevicollis and the origin of metazoans.”  Nature 451:783–788.  2008.  The first genome of a choanoflagellate, from Nicole King's group at the University of California, Berkeley.  It catalogues which protein domains once thought unique to animals are present in a single-celled relative, and which animal signalling pathways and transcription factors are absent.  Mirror:  PMC

1. 59a.

   Section Discussion:  “In contrast, components of most other intercellular signalling pathways, as well as many of the diverse transcription factors that comprise the developmental toolkit of modern animals, are absent.”  Section Results:  “no receptors or ligands were identified from the NHR (nuclear hormone receptor), WNT and TGF-β signalling pathways”. ↩

Schultz, Darrin T. et al.  “Ancient gene linkages support ctenophores as sister to other animals.”  Nature 618(7963):110–117.  2023.  A test of the first branch of animals using blocks of genes that have stayed together on the same chromosomes since before animals, compared across comb jellies, sponges, other animals and single-celled relatives.  Some blocks were fused irreversibly in sponges and the other animals but not in comb jellies, which puts comb jellies first.

1. 60a.

   Section Implications for early animal evolution:  “either complex neural systems arose more than once”.  “or neuronal cell types were present in the metazoan ancestor but were lost in the sponge lineage”. ↩ ↩

Sakarya, Onur et al.  “A post-synaptic scaffold at the origin of the animal kingdom.”  PLoS ONE 2(6):e506.  2007.  A genome search of the sponge Amphimedon queenslandica for 36 families of genes that build the receiving side of a vertebrate synapse, with gene trees and staining of five genes in sponge larvae.  Most were present and are switched on together in one larval cell type, though sponges have no synapses.  The authors say protein-level evidence is still needed.  Mirror:  PMC

1. 61a.

   Section Abstract:  “We show that the genome of the demosponge Amphimedon queenslandica possesses a nearly complete set of post-synaptic protein homologs whose conserved interaction motifs suggest assembly into a complex structure.”  “Among the few post-synaptic genes absent from Amphimedon, but present in Eumetazoa, are receptor genes including the entire ionotropic glutamate receptor family.” ↩

Musser, Jacob M. et al.  “Profiling cellular diversity in sponges informs animal cell type and nervous system evolution.”  Science 374(6568):717–723.  2021.  A whole-body single-cell gene-activity atlas of the freshwater sponge Spongilla lacustris, with electron and x-ray microscopy.  It found 18 cell types, including secretory cells that wrap the feeding cells' projections, and read them as an early signalling system.  One species only.  Mirror:  PMC

1. 62a.

   Section Abstract:  “The evolutionary origin of metazoan cell types such as neurons and muscles is not known.” ↩ ↩

Meyer, Stephen C.  Darwin's Doubt: The Explosive Origin of Animal Life and the Case for Intelligent Design.  HarperOne, New York.  Paperback, with an epilogue answering critics edition.  2014.  The main intelligent-design book on the origin of the animal phyla.  Its chapters 13, 14 and 16 argue from embryology that mutation and selection cannot build body plans:  early-acting mutations are lethal, the core gene networks cannot change, and part of the information for form lies outside DNA.  Meyer directs the Discovery Institute's Center for Science and Culture.

1. 63a.

   Chapter 13, The Origin of Body Plans, section Developmental Gene Regulatory Networks:  “At the center of this regulatory hierarchy are the regulatory networks that specify the axis and global form of the animal body plan during development. These dGRNs cannot vary without causing catastrophic effects to the organism.”  “Indeed, there are no examples of these deeply entrenched, functionally critical circuits varying at all.” ↩ ↩

2. 63b.

   Chapter Epilogue, Responses to Critics of the First Edition, section The Genes in Gene Regulatory Networks Contain Genetic Information:  “Specifically, rewiring a gene regulatory network in the way Marshall envisions would require multiple coordinated mutations in the cis -regulatory regions adjacent to various genes in these regulatory networks.”  “Moreover, producing new functional genes in this manner—that is, as the result of multiple coordinated mutations—would require implausibly long waiting times of the kind discussed in Chapter 12.” ↩

3. 63c.

   Chapter 14, The Epigenetic Revolution, section Neo-Darwinism and the Challenge of Epigenetic Information:  “If DNA isn’t wholly responsible for the way an embryo develops—for body-plan morphogenesis—then DNA sequences can mutate indefinitely and still not produce a new body plan, regardless of the amount of time and the number of mutational trials available to the evolutionary process. Genetic mutations are simply the wrong tool for the job at hand.” ↩

4. 63d.

   Chapter 16, Other Post-Neo-Darwinian Models, section Major But Not Viable, Viable But Not Major:  “As we saw in Chapter 13, the experiments of scientists such as Nüsslein-Volhard and Wieschaus have shown definitively that early-acting body-plan mutations invariably generate embryonic lethals—dead animals incapable of further evolution.” ↩

Hinman, Veronica F., and Eric H. Davidson.  “Evolutionary plasticity of developmental gene regulatory network architecture.”  Proceedings of the National Academy of Sciences 104(49):19404–19409.  2007.  A wider comparison of the same sea urchin and sea star networks.  A five-gene core, which the authors call a kernel, was the same in both;  almost every link outside it differed, including links upstream of the kernel.  Mirror:  PMC

1. 64a.

   Section Abstract:  “We show here that the GRN structure upstream and downstream of the conserved network kernel has, by contrast, diverged extensively.”  Section SI Text:  “The surprising fact is that outside of the kernel not one of the genes included in Fig. 5 has all of the same regulatory inputs in the two GRNs.” ↩ ↩

Israel, Jennifer W. et al.  “Comparative developmental transcriptomics reveals rewiring of a highly conserved gene regulatory network during a major life history switch in the sea urchin genus Heliocidaris.”  PLoS Biology 14(3):e1002391.  2016.  Gene-activity time courses from egg to larva in two sea urchins of one genus, one with a feeding larva and one that develops from a large yolky egg without feeding, plus an outgroup species.  Expression changed most on the branch to the non-feeding species, and most of all inside the well-mapped sea urchin gene network.  The Wray and Raff laboratories have studied this pair for decades.  Mirror:  PMC

1. 65a.

   Section Introduction:  “In particular, key patterning mechanisms such as dorsoventral axis specification, the establishment of the primary signaling center of the embryo, and early cell fate specification differ between the two species.”  “Notably, some of these modifications involve developmental mechanisms that were previously conserved for 10s–100s of millions of years before changing dramatically and rapidly during the evolution of lecithotrophy”.  “(divergence time = 5 Myr)”. ↩

Davidson, Eric H.  “Evolutionary bioscience as regulatory systems biology.”  Developmental Biology 357(1):35–40.  2011.  An essay by the Caltech biologist whose laboratory mapped the sea urchin's embryonic gene networks.  It rejects classical neo-Darwinism as a model for body-plan evolution, explains why modern networks resist change at their upper levels, and proposes that the first networks were shallow enough for selection to shape.  Meyer's chapter 13 quotes it more than any other source.  Mirror:  PMC

1. 66a.

   Section The significance of crown group dGRN design:  “Thus we can think of a crown group dGRN as an evolutionarily terminal, finely divided, extremely elegant control system that allows continuing alteration, variation, and evolutionary experimentation only after the body plan per se has formed, i.e., in structural terms, at the dGRN periphery, and in developmental terms, late in the process.”  “The basic control features of the initial dGRNs of the Precambrian and early Cambrian must have differed in fundamental respects from those now being unraveled in our laboratories. The earliest ones were likely hierarchically shallow rather than deep, so that in the beginning adaptive selection could operate on a larger portion of their linkages.” ↩

2. 66b.

   Section The significance of crown group dGRN design:  “Deconstructing the evolutionary process by which stem group body plans were stepwise formulated will require us to traverse the conceptual pathway to dGRN elegance, beginning where no modern dGRN provides a model.” ↩ ↩

Nüsslein-Volhard, Christiane, and Eric Wieschaus.  “Mutations affecting segment number and polarity in Drosophila.”  Nature 287(5785):795–801.  1980.  The large fruit-fly mutation screen, done at Heidelberg, that found the genes that lay out the larva's segments, work that won the 1995 Nobel Prize in Physiology or Medicine.  It searched specifically for mutations that kill the embryo and change its segment pattern.

1. 67a.

   Page 795, section Abstract:  “In systematic searches for embryonic lethal mutants of Drosophila melanogaster we have identified 15 loci which when mutated alter the segmental pattern of the larva. These loci probably represent the majority of such genes in Drosophila.” ↩ ↩

Durrett, Rick, and Deena Schmidt.  “Waiting for two mutations:  with applications to regulatory sequence evolution and the limits of Darwinian evolution.”  Genetics 180(3):1501–1509.  2008.  A population-genetics model, with simulations, of how long it takes for one transcription-factor binding site to be switched off and another switched on, for fly-sized and human-sized populations.  Two Cornell mathematicians wrote it partly to answer Michael Behe;  Behe replied and the authors conceded one correction in a published exchange.  Mirror:  PMC

1. 68a.

   Page 1501, section Abstract:  “Consistent with recent experimental observations for Drosophila, we find that a few million years is sufficient, but for humans with a much smaller effective population size, this type of change would take >100 million years.” ↩ ↩

Batten, Don.  “Hox (homeobox) genes—evolution's saviour?”  Creation Ministries International.  Feb 11, 2006.  A short web article by a CMI scientist arguing that Hox genes cannot drive evolution because homeotic mutants are malformed.  Its only cited evidence is a 1994 review by the biochemist Christian Schwabe.

1. 69a.

   “Changes to homeotic genes cause monstrosities (two heads, a leg where an eye should be, etc.); they do not change an amphibian into a reptile, for example.” ↩

Amadei, Gianluca et al.  “Embryo model completes gastrulation to neurulation and organogenesis.”  Nature 610(7930):143–153.  2022.  Mouse embryonic stem cells combined with two kinds of supporting stem cells, with no egg or fertilisation, self-organised into embryo-like structures that reached the stage of an 8.5-day mouse embryo.  On average 21% of the structures passed from day 4 to day 5.  The models are not viable embryos.  Mirror:  PMC

1. 70a.

   Section Abstract:  “Our embryo model displays headfolds with defined forebrain and midbrain regions and develops a beating heart-like structure, a trunk comprising a neural tube and somites, a tail bud containing neuromesodermal progenitors, a gut tube, and primordial germ cells.” ↩

The count is the nervous-systems article's own, on the tree of Schultz and colleagues.⁶⁰  With a cost g for each gain of neurons and l for each loss, one origin costs one gain and two losses, and the cheapest two-origin story costs two gains.  So one origin is cheaper only if a loss costs less than half a gain.  No one has measured either cost. ↩

Naor, Adit et al.  “Low species barriers in halophilic archaea and the formation of recombinant hybrids.”  Current Biology 22:1444–1448.  2012.  A laboratory study of cell fusion between two species of the salt-loving archaeon Haloferax, which formed fused cells carrying both genomes and then separated into recombinant hybrids.

1. 72a.

   Section Summary:  “a diploid state containing the full genetic repertoire of both parental cells”. ↩

Smith, F. LaGard.  “Top 3 reasons why the popular evolution story is a myth.”  The Christian Post.  May 17, 2018.  An opinion piece by the author of Darwin's Secret Sex Problem (WestBow, 2018), summarising the book's three arguments against an evolutionary origin of sex.

1. 73a.

   Paragraph after the three problems:  “Taken together, the first two problems are quietly acknowledged by evolutionists to be the "Queen of evolutionary problems" for which, despite their best efforts, they have no answers.” ↩

McLatchie, Jonathan.  “Sex, the Queen of Problems in Evolutionary Biology.”  Science and Culture Today (Discovery Institute).  Jul 13, 2011.  A blog essay by an intelligent-design writer.  It sets out the costs of sex, quotes Bell, Zimmer and Maynard Smith, and argues that neither the origin nor the maintenance of sex is explained by Darwinian evolution.  Mirror:  Evolutionnews.org

1. 74a.

   “Sex is not only unnecessary, but it ought to be a recipe for evolutionary disaster.”  “By all rights, any group of animals that evolves sexual reproduction should be promptly outcompeted by nonsexual ones. And yet sex reigns… Why is sex a success, despite all its disadvantages?” ↩

Zimmer, Carl.  Evolution: The Triumph of an Idea.  Heinemann.  2002.  A popular history of evolutionary biology by a science journalist, written as the companion to the PBS series Evolution.  Its chapter on sex presents the cost problem and then the parasite hypothesis.

1. 75a.

   Page 231:  “Why is sex a success, despite all its disadvantages? Scientists have recently been gathering support for a surprising hypothesis: sex fights off parasites.” ↩

Thomas, Brian.  “Why Do Animals Use Sexual Reproduction?”  Institute for Creation Research.  May 28, 2015.  A news commentary by an ICR science writer on Lumley and colleagues' 2015 flour-beetle experiment.  It accepts that the beetles show a benefit of sexual selection and argues that the deeper reason animals keep reproducing sexually is that their reproductive system cannot change.

1. 76a.

   “Perhaps the reason why these beetles keep on reproducing sexually is not because of the nice benefits of staving off extinction to a future date, but simply because they have no other way to do it!” ↩

Lutes, Aracely A. et al.  “Laboratory synthesis of an independently reproducing vertebrate species.”  Proceedings of the National Academy of Sciences 108(24):9910–9915.  2011.  A laboratory study in which crosses between a sexual male whiptail lizard and a parthenogenetic female produced tetraploid females that went on to reproduce by cloning for several generations.

1. 77a.

   Section Discussion:  “The four lineages described here constitute a laboratory-generated vertebrate species”. ↩

Tomkins, Jeffrey P.  “Algae Multicellular Evolution Study Debunked.”  Institute for Creation Research.  Mar 21, 2019.  A young-earth response to the 2019 predator experiment by a geneticist at the Institute for Creation Research, written from the paper and its press coverage.

1. 78a.

   Fourth paragraph:  “In two of the five isolates, the algae seemed to permanently express the tendency to cluster together. In the genetically diverse populations exposed to the predator, this never happened.” ↩

Herron, Matthew D. et al.  “De novo origins of multicellularity in response to predation.”  Scientific Reports 9:2328.  2019.  A predator experiment in Chlamydomonas.  Five outcrossed populations were exposed to the ciliate Paramecium for 50 weekly transfers, and two evolved heritable clusters that protected them from being eaten;  unexposed controls did not.  The evolved strains were described and tested for survival but not sequenced.  Mirror:  PMC

1. 79a.

   Page 2:  “After 50 weekly transfers (~750 generations), simple multicellular structures evolved in two of five predator-selected populations (B2 and B5).” ↩

Blankenship, Robert E., and A. L. M. Haffa.  “Why Do We Need to Teach the Evolution of Photosynthesis?”  Photosynthesis. Energy from the Sun: 14th International Congress on Photosynthesis.  Springer.  2008.  A conference paper, co-written by a leading photosynthesis researcher, on teaching the evolution of photosynthesis against intelligent-design criticism.

1. 80a.

   Section Abstract:  “Photosynthesis is a process that has been portrayed in ID literature as irreducibly complex in those aspects that are not well understood, and elegant by design in those that are.” ↩

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. 81a.

   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.” ↩

Wood, Johanna.  “Banded Iron Formations: What Are They, and Why Should We Care?”  Proceedings of the 2025 New Scholars International Conference on Creationism.  2025.  A student poster abstract from Cedarville University reviewing recent work on banded iron formations and asking what a biblical model of them would have to explain.

1. 82a.

   Section Abstract:  “Also, issues such as the BIFs’ disappearance in the Phanerozoic, their ambiguous formation mechanisms, microbial roles, and the GOE’s validity in earth history would also need to be addressed in a thorough Biblical model.” ↩

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. 83a.

   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. ↩

Kimura, Motoo, and Takeo Maruyama.  “The mutational load with epistatic gene interactions in fitness.”  Genetics 54(6):1337–1351.  1966.  A theory paper by two founders of neutral and nearly neutral population genetics.  It works out the load of harmful mutations when they interact, for sexual and for asexual populations, and shows that interaction lowers the load only when there is recombination.  Mirror:  PMC

1. 84a.

   Page 1343:  “The above treatment shows that under asexual reproduction, epistasis has no effect in reducing the mutational load.”  Equation 3.3 on the same page gives mean fitness W0 e^(−2M), where 2M is the number of new mutations per individual per generation, written U in the sex article. ↩

Herron, Matthew D. et al.  “Triassic origin and early radiation of multicellular volvocine algae.”  Proceedings of the National Academy of Sciences 106(9):3254–3258.  2009.  A molecular-clock dating of the volvocine algae from four genes, calibrated on a date taken from a broader clock study because the family has no usable fossils.  It moved the origin of Volvox back from 50 to 75 million years to about 234 million years and dated each of Kirk's steps.  The dates are model outputs with wide intervals.  Mirror:  PMC

1. 85a.

   Section Results and Discussion:  “The initial shift from a unicellular to a multicellular lifestyle occurred by 223 Ma (200–248; Fig. 2, node B)”.  “The first step in a reproductive vs. somatic division of labor, the origin of sterile somatic cells, happened between 134 and 227 Ma in the lineage leading to Astrephomene, between 30.7 and 216 Ma in the lineage leading to Volvox barberi and V. globator, and between 76.6 and 116 Ma in the lineage leading to Pleodorina and the remaining species of Volvox.” ↩

Tong, K. et al.  “Genome duplication in a long-term multicellularity evolution experiment.”  Nature 639:691–699.  2025.  The same long-term yeast populations followed to 1,000 days.  The cells doubled their whole genome within 50 days in every population, and later gains and losses of single chromosomes tracked the gain and loss of large size.  The paper revises the 2023 account, which credited point mutations.  Mirror:  PMC

1. 86a.

   Section Results:  “We have continued the MuLTEE for 400 more transfers, or 1,000 days (approximately 5,000 generations) in total”. ↩