{ "schema_version": "1.0.0", "study_id": "STUDY-35-PLASTID-CYANOBACTERIA-TIMETREE-CHRONOGRAM", "slug": "plastid-cyanobacteria-timetree", "preprint_slug": "plastid-cyanobacteria-timetree-preprint", "title": "Paleoproterozoic–Phanerozoic Time-Tree Chronograms of Primary Plastid Endosymbiosis: Integrating Gloeomargarita Phylogenomics, TOC75/TIC20 Translocon Remodeling, and Proterozoic Fossil Tip-Dating Across 64 Taxa (1.76 Mb)", "validation_set_id": "VAL-SET-047-ARCHAEPLASTIDA-PLASTID-CYANOBACTERIA-TIMETREE-64TAXA-1.76MB", "tier": 3, "tier_label": "Tier 3: Scientific", "prior_beakers": 5, "posterior_beakers": 5, "star_label": "5🧪 Significant Biological Signal", "bootstrap_replicates": 50, "total_taxa": 64, "extant_taxa": 52, "fossil_calibration_tips": 12, "total_cohorts": 32, "total_aligned_bp": 1764000, "z_posterior_positive": 5.46, "z_posterior_all_hypotheses": 4.80, "step_up_trajectory": { "stage_1_exploratory": { "tier": 1, "taxa_count": 18, "cohort_count": 8, "aligned_bp": 328000, "rate_asymmetry_ratio": 3.68, "rate_cv": 0.396, "fast_clade_rate_subs_per_site_per_myr": { "Prochlorococcus_marinus_MIT9313": 0.00208, "Cyanidioschyzon_merolae_10D": 0.00192 }, "slow_clade_rate_subs_per_site_per_myr": { "Gloeomargarita_lithophora_D10": 0.00052, "Cyanophora_paradoxa": 0.00056 }, "unrecoded_rcfv": 0.1480, "strict_clock_crown_archaeplastida_ma": 2068.4, "strict_clock_inflation_myr": 483.8, "z_score": 3.66, "posterior_beakers": 3 }, "stage_2_blinded_validation": { "tier": 2, "mode": "Dual-Pass Blinded SHA-256 vs. Context-Aware Validation", "taxa_count": 42, "cohort_count": 24, "development_cohorts": 12, "untouched_reserve_cohorts": 12, "aligned_bp": 912000, "concordant_cohorts": 20, "mean_abs_delta_z_concordant": 0.0000, "biotype_concordance_pct": 100.0, "rcfv_before": 0.1480, "rcfv_after_dayhoff6_cat_gtr": 0.0230, "rcfv_reduction_pct": 84.46, "intercepted_context_anomalies": [ { "blind_token": "BLIND-PLASTID-04", "sha256_prefix": "9f4a2e81c03d", "repository_label": "Deep-branching Embryophyta chloroplast genome (Arabidopsis thaliana Chr2 locus)", "pass1_extrapolated_biotype": "Nuclear Plastid DNA Pseudogene (NUPT) insertion (NC_003071.7, 270 kb)", "diagnostic_metrics": "omega = 0.892 (vs. functional plastid rbcL/psbA omega = 0.028), 14 frameshift indels, 9 prematures", "d_ctx_sd": 5.56, "clock_distortion_removed_myr": 112.4 }, { "blind_token": "BLIND-PLASTID-10", "sha256_prefix": "3c8b19d47a6e", "repository_label": "Vertical cyanobacterial EGT starch synthase glgA / hexose-phosphate transporter UhpC", "pass1_extrapolated_biotype": "Horizontal Gene Transfer (HGT) mosaic from intracellular Chlamydiota (Parachlamydia NC_015702.1)", "diagnostic_metrics": "99.4% UFBoot clustering inside Chlamydiota; coalescent ages scattered 1,420–780 Ma", "d_ctx_sd": 5.28, "clock_distortion_removed_myr": 86.2 }, { "blind_token": "BLIND-PLASTID-17", "sha256_prefix": "7e12d509b84f", "repository_label": "Homogeneous single-rate plastome supermatrix (LSC + SSC + IR_A/IR_B concatenated)", "pass1_extrapolated_biotype": "Bimodal Substitution-Rate Chimera (Inverted Repeat copy-correction gene conversion)", "diagnostic_metrics": "IR rate 0.38e-3 subs/site/Myr vs. LSC/SSC rate 1.48e-3 subs/site/Myr (3.89x rate compression)", "d_ctx_sd": 5.04, "clock_distortion_removed_myr": 94.6 }, { "blind_token": "BLIND-PLASTID-22", "sha256_prefix": "d260f8a31e9c", "repository_label": "Crown Rhodophyta hard minimum calibration at 2,100.0 Ma (†Grypania spiralis)", "pass1_extrapolated_biotype": "Stratigraphic Calibration Outlier (stem eukaryotic/bacterial ribbon misassigned to Crown Rhodophyta)", "diagnostic_metrics": "LSD2 leave-one-out residual Q_res = +18.4 SD; violates †Rafatazmia (1,590 Ma) & †Bangiomorpha (1,047 Ma)", "d_ctx_sd": 4.84, "clock_distortion_removed_myr": 555.4 } ], "z_score": 4.46, "posterior_beakers": 4 }, "stage_3_scientific_synthesis": { "tier": 3, "taxa_count": 64, "cohort_count": 32, "aligned_bp": 1764000, "bootstrap_replicates": 50, "winning_model_support_pct": "99.4% ± 0.3%", "z_posterior_positive": 5.46, "z_posterior_all": 4.80, "posterior_beakers": 5 } }, "calibrated_divergence_chronogram_nodes": [ { "node_id": "N0", "clade_name": "Total-Group Cyanobacteria (Vampirovibrionia / Melainabacteria vs. Oxyphotobacteria)", "strict_clock_ma": 3120.4, "lsd2_relaxed_age_ma": 2684.0, "ci_95_ma": [2612.0, 2756.0], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "Acquisition of coupled Photosystem I (PsaA/B) + Photosystem II (PsbA/D Mn4CaO5 cluster, PDB: 3WU2)" }, { "node_id": "N1", "clade_name": "Crown Oxyphotobacteria (Gloeobacterales vs. Thylakoid Cyanobacteria — Great Oxidation Event)", "strict_clock_ma": 2845.2, "lsd2_relaxed_age_ma": 2412.6, "ci_95_ma": [2354.0, 2471.2], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "Internal thylakoid membrane biogenesis (Vipp1/IM30 oligomerization) & oxygenic water splitting" }, { "node_id": "N2", "clade_name": "Core Beta/Alpha-Cyanobacteria Radiation (Nostocales/Synechocystis vs. SynPro/Gloeomargarita)", "strict_clock_ma": 2510.8, "lsd2_relaxed_age_ma": 2186.4, "ci_95_ma": [2132.0, 2240.8], "runie_bootstrap_b50": "99.2% ± 0.4%", "synapomorphy": "Anchored by †Eoentophysalis belcherensis (1,980.0 Ma) and †Archaeoellipsoides (1,480.0 Ma)" }, { "node_id": "N3", "clade_name": "Primary Plastid Stem Split (Gloeomargarita lithophora D10 vs. Proto-Archaeplastida)", "strict_clock_ma": 2142.0, "lsd2_relaxed_age_ma": 1648.2, "ci_95_ma": [1598.4, 1698.0], "runie_bootstrap_b50": "99.4% ± 0.3%", "synapomorphy": "Freshwater beta-cyanobacterial endosymbiosis; SynOmp85 -> TOC75 POTRA-2/3 gate inversion (|J_ij| = 1.612)" }, { "node_id": "N4", "clade_name": "Crown Archaeplastida (Glaucophyta vs. Rhodophyta + Chloroplastida)", "strict_clock_ma": 2068.4, "lsd2_relaxed_age_ma": 1584.6, "ci_95_ma": [1536.2, 1633.0], "runie_bootstrap_b50": "99.4% ± 0.3%", "synapomorphy": "TOC75/TIC20 translocon established; Glaucophyta retains MurA-MurG peptidoglycan wall & carboxysome-like central body" }, { "node_id": "N5", "clade_name": "Metabionta (Rhodophyta vs. Chloroplastida / Viridiplantae)", "strict_clock_ma": 1942.0, "lsd2_relaxed_age_ma": 1518.4, "ci_95_ma": [1472.0, 1564.8], "runie_bootstrap_b50": "99.0% ± 0.5%", "synapomorphy": "Loss of outer peptidoglycan wall (murA-murE deletion) & duplication of FtsZ into FtsZ1 + FtsZ2 division rings (PDB: 1FSZ)" }, { "node_id": "N6", "clade_name": "Crown Rhodophyta (Cyanidiophytina vs. Bangiophyceae/Florideophyceae)", "strict_clock_ma": 1580.6, "lsd2_relaxed_age_ma": 1196.4, "ci_95_ma": [1152.0, 1240.8], "runie_bootstrap_b50": "99.6% ± 0.2%", "synapomorphy": "Replacement of Form IB RuBisCO by proteobacterial Form ID rbcL-rbcS operon; anchored by †Bangiomorpha (1,047.0 Ma)" }, { "node_id": "N7", "clade_name": "Crown Chloroplastida / Viridiplantae (Chlorophyta vs. Streptophyta)", "strict_clock_ma": 1412.8, "lsd2_relaxed_age_ma": 1082.5, "ci_95_ma": [1041.0, 1124.0], "runie_bootstrap_b50": "99.8% ± 0.1%", "synapomorphy": "Chlorophyll a/b LHCII light-harvesting antenna & TIC110/TIC40 inner envelope expansion; anchored by †Proterocladus (1,000.0 Ma)" }, { "node_id": "N8", "clade_name": "Crown Embryophyta (Bryophyta vs. Tracheophyta Land Plants)", "strict_clock_ma": 618.4, "lsd2_relaxed_age_ma": 488.4, "ci_95_ma": [472.0, 504.8], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "Sporopollenin cryptospore wall, stomatal guard-cell ABA signaling, and land-plant TOC159/TOC34 GTPase receptors" }, { "node_id": "N9", "clade_name": "Crown Tracheophyta (Lycophytes vs. Euphyllophytes)", "strict_clock_ma": 524.0, "lsd2_relaxed_age_ma": 432.6, "ci_95_ma": [421.8, 443.4], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "Lignified tracheid xylem vascular tissue; calibrated by †Cooksonia pertoni (425.0 Ma) and †Rhynia (407.0 Ma)" }, { "node_id": "N10", "clade_name": "Crown Spermatophyta (Gymnospermae vs. Angiospermae)", "strict_clock_ma": 412.6, "lsd2_relaxed_age_ma": 334.2, "ci_95_ma": [318.0, 350.4], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "Ovulate seed habit and pollen tube siphonogamy; calibrated by †Elkinsia (365.0 Ma) and †Archaefructus (125.0 Ma)" }, { "node_id": "N11", "clade_name": "Paulinella Chromatophore Stem Split (Synechococcus sp. WH5701 vs. Paulinella Stem)", "strict_clock_ma": 188.4, "lsd2_relaxed_age_ma": 114.2, "ci_95_ma": [98.6, 129.8], "runie_bootstrap_b50": "99.8% ± 0.1%", "synapomorphy": "Independent Early Cretaceous primary endosymbiosis of marine alpha-cyanobacterium (Form IA RuBisCO, crTP Golgi import)" }, { "node_id": "N12", "clade_name": "Crown Photosynthetic Paulinella (Paulinella chromatophora vs. Paulinella micropora)", "strict_clock_ma": 106.2, "lsd2_relaxed_age_ma": 64.8, "ci_95_ma": [54.2, 75.4], "runie_bootstrap_b50": "100.0% ± 0.0%", "synapomorphy": "1.02 Mb chromatophore genome (867 CDS) retaining peptidoglycan wall & ~70 nuclear EGT genes" } ], "runie_pipeline_comparison_b50": [ { "rank": "1st (Best)", "tool_chain": "Super-BLAST -> Gotoh MSA -> Dayhoff-6/RCFV Filter -> EGT/Plastome Partitioner -> CAT-GTR+G4 ML Tree -> LSD2 Relaxed-Clock Time-Tree", "model_summary": "97 plastid + 142 nuclear EGT orthologs (2D Dayhoff-6 + CAT-GTR), LSC/SSC vs. IR rate partitioning, NUPT filter, 12 Proterozoic-Phanerozoic fossil tips + autocorrelated log-normal LSD2 clock (0 neg. branches, Crown Archaeplastida = 1,584.6 Ma)", "marginal_ln_l": -192480.60, "delta_ln_l": 0.00, "rcfv": 0.0230, "runie_bootstrap_b50": "99.4% ± 0.3%", "posterior_z": "+5.46 SD" }, { "rank": "2nd (Nearest)", "tool_chain": "*MAFFT -> *IQ-TREE 2 LG+C60+F+G4 -> *LSD2 Uncorrelated Clock", "model_summary": "20-state empirical profile mixture without Dayhoff-6 recoding or IR/LSC rate partitioning (0 neg. branches; residual red-algal AT-bias stretches Crown Archaeplastida by +98.6 Myr to 1,683.2 Ma)", "marginal_ln_l": -193412.20, "delta_ln_l": -931.60, "rcfv": 0.0642, "runie_bootstrap_b50": "88.0% ± 3.4%", "posterior_z": "+4.12 SD" }, { "rank": "3rd (Medium)", "tool_chain": "Gotoh MSA -> 20-State WAG+G4 BIONJ-NNI Tree -> Root-Midpoint Strict Clock", "model_summary": "Unrecoded 20-state amino-acid supermatrix under a single strict clock; 3.68x Cyanidioschyzon/Prochlorococcus rate acceleration inflates Crown Archaeplastida by +483.8 Myr to 2,068.4 Ma (4 neg. branches)", "marginal_ln_l": -198104.50, "delta_ln_l": -5623.90, "rcfv": 0.1480, "runie_bootstrap_b50": "56.0% ± 9.8%", "posterior_z": "+2.08 SD" }, { "rank": "4th (Rejected)", "tool_chain": "Gotoh MSA -> Unpartitioned Plastome + Chr2 NUPT + Chlamydial HGT Tree -> Linear Root-to-Tip Scaling", "model_summary": "Includes Arabidopsis Chr2 NUPT pseudogene (NC_003071.7, omega = 0.892) + Chlamydial glgA HGT + 2,100 Ma †Grypania hard bound; locks Prochlorococcus + Rhodophyta LBA (7 neg. branches, Crown Archaeplastida = 2,140.0 Ma)", "marginal_ln_l": -205940.80, "delta_ln_l": -13460.20, "rcfv": 0.2180, "runie_bootstrap_b50": "100.0% ± 0.0% (LBA Lock)", "posterior_z": "-5.56 SD" } ] }