{"paper_id":"1ff6cc1e-1e92-4863-8370-7945942e61a2","body_text":"A ribozyme mass extinction at the RNA-cellular \nboundary and its potential imprint on the genetic code \nIdo Bachelet \nThe Scojen Institute for Synthetic Biology, Reichman University, Herzliya, Israel \nEmail for correspondence: ido.bachelet@post.runi.ac.il \n \nAbstract \nThe transition from the RNA world to cellular life remains one of the least understood events in \nthe history of biology. This study proposes that geochemical changes approximately 3.9-3.8 \nbillion years ago triggered a mass extinction of ribozymes - possibly the first extinction event on \nEarth - and that the genetic code bears the fingerprints of its survivors. The argument draws on \nparallels with animal mass extinctions, where generalist species survive, dominate \npost-extinction ecosystems as disaster taxa, and shape subsequent evolutionary trajectories. \nAmong small self-cleaving ribozymes, the hammerhead ribozyme accounts for approximately \n91% of all ~221,000 known sequences and is the only ribozyme that is found across all \nkingdoms of life. The phylogeny of hammerhead ribozymes exhibits a star-like topology \nconsistent with rapid post-bottleneck expansion, and its small size, ability to tolerate diverse \nchemical conditions, and broad substrate specificity suggest it was a resilient generalist feeder \nfitting as a disaster taxon. Other families of ribozymes possibly survived in small, taxonomically \nisolated populations resembling relict species in ecological refugia. It is suggested that the body \nplans of surviving ribozymes seeded the primitive processes that would later become the genetic \ncode, for example with RNA-degrading trinucleotides becoming stop codons, partitioning the \ntrinucleotide space into signals of termination and translation. This hypothesis proposes a \nreframing of the origin of the genetic code in part as an ecological legacy rather than a purely \nchemical inevitability.  \n \n \n1 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nIntroduction \nThe RNA world hypothesis posits a period in Earth's early history during which RNA molecules \nserved simultaneously as genetic material and catalytic agents 1,2. This hypothesis, grounded in \nthe discovery of catalytic RNA 3,4, is now broadly accepted as a foundation of origin-of-life \ntheory. Yet the transition from the RNA world to the cellular world remains one of the least \nunderstood events in the history of life 5,6. \nMost accounts of this transition emphasize its constructive aspects: the synthesis of fatty acid \nvesicles, the encapsulation of replicating molecules, and the emergence of protocells with \nrudimentary metabolism 7-9. Less discussed is the possibility that this transition was also \ndestructive - that the geochemical changes enabling cellular life simultaneously devastated the \npre-existing RNA ecosystem. In the animal kingdom, the most consequential evolutionary \ntransitions are routinely associated with mass extinction events 10,11. The end-Permian extinction \ncleared the way for dinosaur radiation 12; the end-Cretaceous extinction enabled the rise of \nmammals 12,13. In each case, what appears as a creative explosion was preceded by catastrophic \ndestruction. \nThis paper proposes that the RNA-to-cellular transition was triggered by a mass extinction of \nribozymes - potentially the first mass extinction event on Earth - and that the genetic code bears \nthe chemical fingerprints of its dominant survivor. The argument proceeds in three steps. First, \ngeochemical evidence is reviewed for environmental changes ~3.9-3.8 Ga that would have been \ncatastrophic for free-living RNA organisms. Second, evidence is presented that extant ribozyme \nfamilies exhibit every hallmark of a post-mass-extinction ecosystem, with the hammerhead \nribozyme as the disaster taxon. Third, computational analysis demonstrates that the trinucleotide \ncomposition of the ribozyme body plan regions maps onto the functional architecture of the \ngenetic code. \n \n \n2 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nResults \nTo identify candidate triggers for a possible major ecological shift occurring at the RNA-cellular \n(R-C) boundary, we surveyed the geochemical literature for changes proposed to have taken \nplace between approximately 4.2 and 3.5 Ga. Four interlinked factors converge within the \ninterval ~3.9-3.8 Ga (Fig. 1). The upper bound of this interval is constrained by geochemical \nmodeling showing the onset of rapid environmental change, and the lower bound by the earliest \nisotopic signatures of biological carbon fixation at ~3.8 Ga 14,15, indicating that cellular life was \nalready emerging by that time. First, the late accretion bombardment declined sharply across this \ninterval 16 (Fig. 1A). While the waning of impact sterilization is typically viewed as permissive \nfor life, it simultaneously reduced the meteoritic delivery of metals and reduced phosphorus \nspecies that had sustained prebiotic chemistry 17,18. Second, surface temperatures plunged from \nover 200°C to near-modern values across this interval (Fig. 1B). While the cooling itself would \nhave expanded the thermal range habitable by RNA, it accelerated silicate weathering of the \nevolving crust, releasing cations into the ocean and drawing down atmospheric CO₂ - directly \ndriving a rapid pH rise 19,20. Ocean pH rose from approximately 5.0 toward near-neutral values \nmore rapidly than previously estimated 21 (Fig. 1C). The sensitivity of RNA to alkaline \nhydrolysis is intrinsic to its chemistry 22, hence rising pH would have progressively shortened the \nhalf-life of exposed ribozymes. Fourth, the delivery of reduced phosphorus by late accretion \ndeclined by orders of magnitude across this interval 17,23 (Fig. 1D), while phosphate sequestration \nthrough mineral precipitation further constrained its availability 18,24. Ribozyme populations thus \nfaced a growing scarcity of their fundamental building blocks. These four stressors are not \nindependent: the declining bombardment drove the thermal crash, which in turn accelerated pH \nequilibration through enhanced weathering, which promoted phosphate mineralization. The \nresult was a synergistic collapse - ribozymes simultaneously lost structural stability, catalytic \ncompetence, and RNA building blocks (food) for reproduction. This convergence of stressors \nmirrors the multi-causal structure of animal mass extinctions, in which synergistic interactions \nbetween environmental changes amplify extinction severity beyond what any single trigger could \nproduce 25,26.  \nA key detail in understanding the R-C transition is the distribution of extant ribozyme families, \nwhich exhibits a pattern strikingly consistent with a post-mass-extinction ecosystem (Fig. 2), \nwith each hallmark finding a direct parallel in the animal mass extinction record (Table 1). Of \napproximately 221,000 known self-cleaving ribozyme sequences across all ten families, \napproximately 199,000 (91%) are hammerhead ribozymes 27,28 (Fig. 2A), a degree of numerical \ndominance characteristic of disaster taxa 29, exceeding even the post-Permian dominance of \nLystrosaurus (70-95% of terrestrial vertebrate individuals) 30,31. The other types of small, \nself-cleaving ribozymes exist in taxonomically isolated populations (82 to ~11,500 sequences) \nconsistent with relict species in ecological refugia 32,33. The hatchet ribozyme (~166 copies \nrestricted entirely to Crassvirales phage genomes) represents an extreme case of refugial \nsurvival, paralleling the coelacanth 34. The hammerhead satisfies many criteria of a disaster \ntaxon: it is a small, adaptive (modular) 35 and versatile ribozyme; it can tolerate and operate in \ndiverse, non-standard chemical conditions 36,37;  and it possesses the broadest substrate specificity \n(NUH↓, covering 12 of 16 dinucleotide combinations) of all small, self-cleaving ribozymes.  \nDisaster taxa expand in a typical pattern. In order to examine whether hammerhead ribozymes \nexhibit such a pattern, the phylogeny of 80,548 unique complete hammerhead sequences \n3 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nspanning 97 genera was constructed. This phylogeny exhibits a star-like topology consistent with \nrapid post-bottleneck expansion (Fig. 2B). Population genetics statistics computed on the full \nstratified subsample (n = 5,036; S = 40) reveal Tajima's D = +3.79 and Fu's Fs = −690.8; \ncoalescent null testing on a matched subsample (n = 200; S = 37; 10,000 replicates) confirms \nboth values are extreme (D = +2.28, upper-tail p = 0.013; Fs = −618.0, p < 0.0001) (Fig. 2C). \nThe discordance between positive D (reflecting deep hierarchical structure across 97 genera) and \nextremely negative Fs (reflecting haplotype excess from rapid within-lineage expansion) is \ndiagnostic of ancient divergence preserved through a bottleneck, followed by explosive radiation \nwithin surviving host lineages 38. \nThe mass extinction of ribozymes placed the hammerheads that survived it at a unique position \nthat allowed these ribozymes to shape the early exit from a pre-code world. The emergence of \nthe genetic code is one of the most profound questions in biology, and is still under active study \n39-42. It has been addressed by various angles, including chemistry and information theory, but \nrarely as a legacy of ecological circumstance at the R-C boundary. Some of the predictions of \nthis hypothesis were next tested. If the hammerhead's body plan shaped part of the emergence of \nthe genetic code, it would have been likely that the functional organs of the hammerhead \npartitioned the trinucleotide sequence space in ways corresponding to functional categories of the \ngenetic code.  \nWe first analyzed stem and core regions from the entire curated dataset of hammerhead \nribozymes to see if such partitions exist. Two results confirmed this hypothesis. Stems and cores \ncontain RNA triplets that clearly segregate by their roles in the genetic code (Fig. 3A). In \nparticular, amino acids that are hypothesized to have entered the genetic code early, are enriched \n2.8-fold in stems (0.8-0.9-fold in cores) (Fig. 3B). A Spearman correlation between codon-level \nstem enrichment and amino acid chronological rank yielded ρ = −0.243 (p = 0.06, permutation p \n= 0.056): marginal, but in the predicted direction. The correlation is driven by the binary \npartition between core-resident and non-core trinucleotides rather than a smooth gradient, \nreflecting the discrete nature of ribozyme architecture.  \nInterestingly, standard stop codons are enriched 3.4-fold in catalytic cores of all ribozymes, and \n6.1-fold in the hammerhead core specifically, while being depleted 0.4-fold in stem regions (Fig. \n3A, Fig. S1). The rationale for catalytically-active RNA triplets assuming the role of stop signals \nis twofold: first, they are hazardous to the integrity (and thus reliability) of RNA messages, so \nthey need to be kept as far as possible from the message (at its end); and second, they are by \ndefinition natural terminators of RNA strands.  \nIn a wide range of hammerhead ribozymes, the most striking feature of the catalytic core is the \nmotif CUGAUGA, which includes two tandem repeats of the UGA (opal/umber) stop codon. \nThe hammerhead is the only of the small self-cleaving ribozymes to contain two stop codons. \nThe unique placement of the hammerhead in the post-extinction world raises the hypothesis that \nUGA was the first primitive stop codon, and was replaced only later by UAA. UGA's leakiness \nand frequent reassignments 43,44 could be reinterpreted not as recent recruitment but as evidence \nof its pre-translational origin; it was never optimized for the release factor system. This is \nsupported by analysis of 492 universally conserved genes across three organisms: universal \ngenes strongly prefer UAA (e.g., E. coli: 88% vs. 64% background, p = 5.4 × 10⁻⁶ ), consistent \nwith UAA as the ancestral translational stop. In S. cerevisiae and H. sapiens, UGA shows less \n4 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\ndownstream context bias than UAA (S. cerevisiae : mean hexamer IC = 0.12 vs. 0.19 bits; H. \nsapiens: 0.06 vs. 0.06 bits), consistent with UGA's function originating in a pre-translational \nsystem where downstream context was irrelevant 45,46 (Fig. 3C, 3D) Furthermore, UGA \nreadthrough sites - where UGA is decoded as selenocysteine or other amino acids rather than \nfunctioning as a stop - show significantly more stable local RNA structure than efficiently \nterminating UGA controls (MFE: −30.6 vs. −16.8 kcal/mol, p = 1.2 × 10⁻⁵ ) and more stem-loop \nstructures (p = 0.006), consistent with structural elements shielding UGA from the termination \nmachinery and preserving its pre-translational accessibility to alternative decoding (Fig. 3E). \n \n \n5 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nDiscussion \nThe central claim of this paper is that the transition from the RNA world to cellular life was \ntriggered by a catastrophic event leading to mass extinction of ribozymes, and that the survivors \nof this event had left a legible imprint on the genetic code. The evidence presented - geochemical \nconvergence of stressors, ecological signatures of mass extinction among extant ribozymes, and \nthe correspondence between the ribozyme body plan and genetic code primitives - lends support \nto this hypothesis.  \nSeveral aspects of this framework warrant further discussion. The mass extinction model makes \na specific prediction about the nature of the geochemical trigger: it should have been rapid \nrelative to evolutionary timescales, multi-causal rather than attributable to a single stressor, and \nselectively destructive of ecologically specialized forms. The convergence of reduced \nbombardment, temperature shift, pH changes, and depletion of available phosphate documented \nfor the 3.9-3.8 Ga interval satisfies all three criteria. While individual parameters remain subject \nto considerable uncertainty - early ocean pH estimates range from 5 to 7 depending on the model \n[Guo & Korenaga, 2025; Krissansen-Totton et al., 2018; Zahnle et al., 2010] - the convergence \nof multiple independent environmental shifts is robust across models. \nThe distinction between ecological stop (UGA) and translational stop (UAA) proposes a \nreframing of the traditional view of UGA as a late addition to the code 47. The present model \npictures UGA as the primary weapon carried by the ribozymes that survived the extinction, who \ncarried it in two copies each; it was in this capacity as a ribozyme-degrading blade, that UGA \nfunctioned as a natural termination signal for any primordial translation process that utilized \nRNA as its message and occurred in its vicinity. This might be a reason why the first amino acids \nto be translated by RNA were translated at the stem, physically remote from this hazardous \ntriplet.  The apparent unreliability of UGA in the modern code reflects its ancestry - a signal \nfrom a fundamentally different world, which was never fully adapted to the modern system it \nwas grandfathered into. \nA further prediction - that UGA readthrough sites would retain structural signatures of their \npre-translational history - was confirmed: readthrough sites show significantly more stable local \nRNA structure than termination controls (p = 1.2 × 10⁻⁵ ) and more stem-loop structures (p = \n0.006). This is consistent with a model in which UGA's transition from ecological stop to \ntranslational stop was completed most efficiently at structurally accessible sites, where release \nfactors could recognize UGA without steric interference. At readthrough sites, elaborate local \nstructures (SECIS elements, viral pseudoknots) shield UGA from the termination machinery, \npreserving its availability for alternative decoding - an echo of the post-extinction world in which \nUGA first acquired biological meaning. \nFinally, the present study raises the possibility of a mass extinction event at the R-C boundary, \nshowing multiple parallels to other extinction events from the geological past. Such catastrophic \nevents are known to not only exterminate many clades, but also open new opportunities and \ntrigger rapid evolution and expansion of new branches. It is intriguing to wonder whether, just as \nthe Cambrian explosion followed the Ediacaran extinction, and the mammalian radiation \nfollowed the dinosaur extinction, the emergence of cellular life and the genetic code followed the \n6 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nextinction of ribozymes, of which we might learn from their fossil record within genomes across \nall kingdoms of life.  \n \n \n7 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nMethods \nTrinucleotide enrichment analysis. Trinucleotide frequencies were computed for catalytic core \nand structural stem regions across all seven small self-cleaving ribozyme families using Rfam \nseed alignments [Kalvari et al., 2021]. Each sequence was parsed into core and stem regions \nbased on published secondary structure annotations. Enrichment was calculated as observed \nfrequency divided by expected frequency (background trinucleotide composition of the full \nsequence). A total of 524,824 stem trinucleotides and 563,867 core trinucleotides were analyzed. \nPyrimidine-pyrimidine dinucleotide analysis. For each ribozyme family, the frequency of \npyrimidine-pyrimidine dinucleotides (CC, CT, TC, TT/UU) was computed in catalytic core \nsequences and compared to the expected frequency of 0.25 under random composition. \nTrifonov consensus chronology correlation. Amino acid chronological ranks were taken from \nTrifonov's consensus chronology [Trifonov, 2004]. For each of the 61 sense codons, stem \nenrichment was calculated as described above. Spearman rank correlation was computed \nbetween codon-level stem enrichment and the chronological rank of the corresponding amino \nacid. Permutation testing (10,000 permutations) was used to assess significance. \nPhylogenetic analysis. 80,548 unique complete hammerhead ribozyme sequences were retrieved \nfrom Rfam [Kalvari et al., 2021] and aligned using MAFFT v7 [Katoh & Standley, 2013]. An \napproximate maximum-likelihood tree was constructed using FastTree 2 [Price et al., 2010] with \nthe GTR+CAT model. \nPopulation genetics statistics. Tajima's D [Tajima, 1989] and Fu's Fs [Fu, 1997] were computed \non a stratified subsample of 5,036 sequences preserving the taxonomic distribution of the full \ndataset (97 genera). The subsample contained 40 segregating sites and 4,555 distinct haplotypes. \nP-values were assessed by coalescent simulation (10,000 replicates). \nUGA contextual analysis. Stop codon usage and flanking nucleotide context were analyzed in \n492 universally conserved genes (ribosomal proteins, translation factors, RNA polymerase \nsubunits) across E. coli (93 genes), S. cerevisiae  (300 genes), and H. sapiens (99 genes), \ncompared against 10,298 total coding sequences. Information content was computed for the six \npositions downstream of each stop codon as IC = Σ pᵢ log₂(pᵢ/0.25). \nUGA readthrough analysis. 25 known UGA readthrough sites (20 human selenoprotein SECIS \nelements: GPX1-4,6, TXNRD1-3, DIO1-3, SELENOP,W,H,I,K,N,S,T, MSRB1; plus 5 cellular \nreadthrough genes: rabbit beta-globin, TMV , MuLV , Sindbis, VEGFA-Ax) were compared \nagainst 26 control UGA terminators from housekeeping genes (8 E. coli, 4 S. cerevisiae, 14 H. \nsapiens). All sequences were fetched from NCBI. Minimum free energy, base-pairing \nprobability, and hairpin loop count were computed using RNAfold from ViennaRNA 2.7.0 \n[Lorenz et al., 2011] on 103-nucleotide windows (50 nt upstream + UGA + 50 nt downstream). \nStatistical comparisons used Mann-Whitney U tests (MFE, pairing probability, hairpin count) \nand Fisher's exact test (UGA paired/unpaired classification). \n8 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nBody plan classification. Ribozyme body plan classification follows the framework established \nin Bachelet [2026], with structural regions designated as bodies (paired stems), cavities (catalytic \ncores), and limbs (unpaired substrate-interacting strands). \n \nAuthor contributions \nI.B. designed experiments, performed experiments, analyzed data, and wrote manuscript. \n \nCompeting interests declaration \nThe author declares no competing interests.  \n \n \n9 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nReferences \n1. Neveu, M., Kim, H.-J. & Benner, S. A. The ‘strong’ RNA world hypothesis: fifty years old. \nAstrobiology 13, 391-403 (2013). \n2. Gilbert, W. Origin of life: The RNA world. 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It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\n44. Böck, A. et al. Selenocysteine: the 21st amino acid. Mol Microbiol 5, 515-520 (1991). \n45. Cridge, A. G., Crowe-McAuliffe, C., Mathew, S. F. & Tate, W. P. Eukaryotic translational \ntermination efficiency is influenced by the 3’ nucleotides within the ribosomal mRNA \nchannel. Nucleic Acids Res 46, 1927-1944 (2018). \n46. Poole, E. S., Brown, C. M. & Tate, W. P. The identity of the base following the stop codon \ndetermines the efficiency of in vivo translational termination in Escherichia coli. EMBO J \n14, 151-158 (1995). \n47. Koonin, E. V . & Novozhilov, A. S. Origin and evolution of the genetic code: the universal \nenigma. IUBMB Life 61, 99-111 (2009). \n \n \n12 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nFigure 1 \n \n \nFigure 1. Geochemical changes at the RNA-cellular boundary. Composite timeline of \nenvironmental variables across the 4.5-3.5 Ga interval, with the proposed R-C boundary interval \n(~3.9-3.8 Ga) indicated. (A) Declining impact flux (data from Bottke & Norman, 2017). (B) \nSurface temperature evolution showing the crash from >200 °C to near-modern values (data \nfrom Guo & Korenaga, 2025). (C) Ocean pH rise from acidic to neutral (data from Guo & \nKorenaga, 2025). (D) Declining delivery of reduced phosphorus by late accretion (data from \nRitson et al., 2020). Together, these panels document a convergence of environmental stressors - \nphysical (bombardment), thermal (cooling), chemical (pH shift), and nutritional (phosphate \ndepletion) - coinciding with the proposed extinction interval. \n \n \n \n \n \n13 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nFigure 2 \n \n \nFigure 2. Evidence for ribozyme mass extinction. (A) Abundance distribution across all ten \nknown small self-cleaving ribozyme families, showing the hammerhead's 91% dominance, a \npattern characteristic of disaster taxa following mass extinction events. (B) Circular phylogeny \nof 80,548 unique hammerhead sequences colored by host taxonomic group. The star-like \ntopology is consistent with post-bottleneck radiation, and the interspersion of distantly related \nhost phyla around the tree reflects repeated independent colonization events rather than \nco-speciation, consistent with horizontal transfer of a single dominant survivor lineage. (C) Null \ndistribution tests for Tajima's D (left) and Fu's Fs (right), each based on 10,000 coalescent \nreplicates (n = 200, S = 40). Blue and orange histograms show the null distributions under \nneutral expectations; red dashed lines indicate the observed values (D = +2.28, upper-tail p = \n0.013; Fs = −618.0, p < 0.0001). Observed D falls in the extreme upper tail (98.7% of null \nreplicates below observed), indicating deep hierarchical structure; observed Fs falls far below \nany null replicate, indicating massive haplotype excess. The discordance between positive D and \nextremely negative Fs is diagnostic of ancient divergence preserved through a bottleneck \nfollowed by rapid within-lineage expansion. Full-sample values (n = 5,036) are D = +3.79, Fs = \n−690.8. \n \n \n14 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nFigure 3 \n \n \nFigure 3. The body plan's imprint on the genetic code. (A) Trinucleotide enrichment heatmap \nshowing stop codons enriched in catalytic cores (6.1-fold) and early amino acid codons enriched \nin structural stems (2.8-fold for Val/Asp/Gly). (B) Stem enrichment vs. amino acid chronological \nrank (Trifonov consensus), showing a marginal but predicted negative correlation at codon level \n(ρ = −0.243, p = 0.06, permutation p = 0.056). (C) Stop codon usage and downstream context in \nuniversally conserved genes across E. coli, S. cerevisiae, and H. sapiens. Left: fractional usage of \nUAA, UAG, and UGA, showing a progressive shift from UAA dominance in E. coli (88% UAA) \nto UGA dominance in H. sapiens (51% UGA), consistent with UGA's gradual co-option from a \npre-translational cleavage signal into a translational stop codon. (D) Mean information content \n(IC) of the downstream hexamer for UAA- vs. UGA-terminated universal genes, showing that \nUAA-terminated genes carry higher downstream sequence constraint in E. coli and S. cerevisiae , \nconsistent with UAA having been under translational selection longer than UGA. (E) UGA \nreadthrough vs. termination structural analysis. Readthrough sites (n = 25; 20 selenoprotein \nSECIS elements, 5 cellular readthrough genes) show significantly more stable local RNA \nstructure than UGA termination controls (n = 26; MFE: −30.6 vs. −16.8 kcal/mol, p = 1.2 × 10⁻⁵ ) \nand more stem-loop structures (2.48 vs. 1.85 hairpins, p = 0.006), consistent with local structure \nshielding UGA from release factors and preserving its accessibility to alternative decoding \nmachinery. \n15 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nTable 1. Parallels between ribozyme and animal mass extinction signatures. Each row identifies \na hallmark signature of mass extinction and recovery, with the ribozyme evidence from this \nstudy and its closest paleontological analog. Ribozyme citations refer to independent published \nstudies except where noted; paleontological analogs span the end-Permian, end-Cretaceous, and \nLate Ordovician mass extinctions. See Supplementary Note S2 for expanded discussion of each \nparallel. \n \nExtinction signature Ribozyme extinction Paleontological analog \nDisaster taxon \ndominance \nHammerhead: ~91% of ~221,000 \nsequences; present in all domains of \nlife \nDe la Peña & García-Robles 2010; \nPerreault et al. 2011 \nLystrosaurus: 70-95% of Early \nTriassic terrestrial vertebrate \nindividuals \nBotha-Brink et al. 2016; Kauffman & \nHarries 1996 \nRelict in \nrefugium \nHatchet: ~166 copies restricted to \nCrassvirales phage genomes \nRfam RF02678; Weinberg et al. 2019 \nLatimeria (coelacanth): 2 species \nin deep-sea caves; presumed \nextinct 66 Myr \nSmith 1939; Amemiya et al. 2013 \nRelict survivor \n(restricted range) \nHairpin (~139), Pistol (~103), \nTwister sister (~129): each in \nnarrow host ranges \nRfam 15.1; Weinberg et al. 2019 \nNautilus: sole extant nautiloid; \nrestricted Indo-Pacific after \nammonite extinction \nWard 1987 \nGeneralist survival \nadvantage \nHammerhead NUH↓: 12/16 \ndinucleotide targets (NHH rule); \ntolerates clay, Fe²⁺, extreme \nconditions \nKore et al. 1998; Athavale et al. 2012; \nBiondi et al. 2007 \nK-Pg bivalves: genera in ≥3 \nbiogeographic provinces had \n~20% extinction vs. 70% for \nrestricted \nJablonski 2005 \nMarine disaster \ntaxa \nHammerhead dominant across \nmarine invertebrate host phyla \n(Trematoda, Bivalvia, \nCephalopoda) \nDe la Peña & García-Robles 2010; \nPerreault et al. 2011 \nClaraia, Unionites, Lingularia: \nlow-diversity high-dominance \nEarly Triassic benthos \nClapham et al. 2016 \nStar phylogeny / \npost-bottleneck \nradiation \nHammerhead: star-like tree across \n97 genera; D = +3.79, Fs = −690.8 \nThis study \nNeoaves: explosive \ndiversification at K-Pg; \nunresolved star polytomy in \nmolecular phylogenies \nJarvis et al. 2014 \n16 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint \n\nFigure S1 \n \n \nFigure S1. Partitioning of amino acid roles and enrichment of stop codons in catalytic cores \nacross small, self-cleaving ribozymes (~ 221,000).  \n17 \n.CC-BY 4.0 International licenseavailable under a \n(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made \nThe copyright holder for this preprintthis version posted March 9, 2026. ; https://doi.org/10.64898/2026.03.05.709948doi: bioRxiv preprint","source_license":"CC-BY-4.0","license_restricted":false}