Permo-Carboniferous–Cenozoic Lepidoptera Genomic Radiation, Angiosperm-Coupled Ditrysia & Papilionoidea Time-Tree, and COX1 DNA Barcode Resolution vs. Deep-Node Saturation Boundaries

Across 92 NCBI RefSeq and GenBank sequence records spanning 1,078,183 bp (1.08 Mb) —comprising 72 complete mitogenomes (1,053,261 bp), 56 profile-anchored 450 bp (150-codon) in-frame COX1 barcode alignments, 15 nuclear coding orthologs ( EF-1α , CAD , CYP6B1 / CYP6B4 ; 20,330 bp), and 16 fossil calibrations ( B = 500 RuNIE replicates)—we resolve the 298.6 Ma Permo-Carboniferous–Cenozoic radiation of Lepidoptera and map the empirical diagnostic boundary between species-level COX1 DNA barcoding and deep-node phylogenomics. While COX1 resolves sister species below 35 Ma ( Ostrinia nubilalis vs. O. furnacalis d K2P = 0.0045; Bombyx mori vs. B. mandarina d K2P = 0.0181, Ti/Tv = 8.00; Papilio machaon vs. P. xuthus d K2P = 0.0651; 2nd-codon H pos2 = 0.1575 bits vs. 3rd-codon H pos3 = 1.1371 bits), 3rd-codon AT bias (82.90% core AT3, RCFV = 0.2845) and transition saturation (Ti/Tv = 0.41–0.73) collapse unpartitioned trees above 85 Ma—a boundary rescued by nuclear coding orthologs, Dayhoff-6 recoding, and Dual-Pass Blinded screening.

Permian–Cenozoic Hymenoptera Genomic Radiation, Orussidae Ectoparasitoid Sisterhood to Apocrita, and the 284–0 Ma Total-Evidence Time-Tree of Parasitoidism and Independent Eusociality

Across 108 NCBI RefSeq sequence records totaling 1,223,380 bp raw (1,173,240 bp aligned supermatrix: 68 complete mitogenomes, 52 in-frame COX1 coding cores, and 40 nuclear Orco/Vg/Vg-R/Dnmt3/MRJP1/Art3 orthologs + 285 Mkv characters; B = 500 RuNIE replicates), our hybrid RuNIE + *MAFFT-L-INS-i + *IQ-TREE2-LG-C60-F-G4 synthesis resolves the 281.4 Ma radiation of Hymenoptera, confirms a single 226.4 Ma origin of parasitoidism uniting Orussidae + Apocrita, unites ants and bees at 148.6 Ma, and maps four independent Cretaceous origins of eusociality.

Carboniferous–Cenozoic Synapsid-to-Mammaliaformes Stem Transition, Detached Three-Bone Middle Ear Evolution, and the 318–0 Ma Crown Mammalia Total-Evidence Time-Tree

A 66-taxon, 1.82 Mb Dual-Pass Blinded Dayhoff-6 + CAT-GTR + Partitioned Mkv + Fossilized Birth-Death synthesis (B = 500 RuNIE replicates) traces the 318 Myr synapsid-to-mammal transition, places Triassic Haramiyida on the mammaliaform stem (214.6 Ma), dates Crown Mammalia to 184.6 Ma and Crown Theria to 158.4 Ma, and resolves the co-evolution of the detached 3-bone middle ear and casein lactation.

Devonian Sarcopterygian Fin-to-Limb Transition, Dipnoi Sisterhood, and the 440–0 Ma Crown Tetrapoda Total-Evidence Time-Tree Chronogram

A 64-taxon, 1.78 Mb Dual-Pass Blinded Dayhoff-6 + CAT-GTR + Partitioned Mkv + Fossilized Birth-Death synthesis resolves Dipnoi (lungfishes) as the 99.6%-supported sister clade to Tetrapoda at 418.4 Ma, traces the Devonian fin-to-limb transition across elpistostegalians (383.8 Ma) and polydactylous stegocephalians (373.4 Ma), and dates Crown Tetrapoda to 346.2 Ma.

Paleoproterozoic Primary Plastid Endosymbiosis, Basal Gloeomargarita Sisterhood, and the 1,648–0 Ma Archaeplastida vs. Paulinella Time-Tree Chronogram

A 64-taxon, 1.76 Mb Dual-Pass Blinded Dayhoff-6 + CAT-GTR + Fossilized Birth-Death synthesis resolves the primary plastid stem divergence from basal freshwater Gloeomargarita lithophora D10 to 1,648.2 Ma, Crown Archaeplastida to 1,584.6 Ma, and the independent Paulinella chromatophora endosymbiosis to 114.2 Ma.

The Metazoan Cambrian Explosion Tree: Total-Evidence Phylogenomics, Hox/miRNA Macrosynteny, and Ediacaran–Cambrian Fossil Tip-Dating Across 68 Extant and Fossil Taxa (1.84 Mb)

A single-column phylogenomic and paleontological preprint monograph synthesizing 68 extant (54) and Ediacaran–Cambrian Lagerstätte fossil (14) metazoan and holozoan outgroup taxa (1.84 Mb + 312 Lewis Mkv+G4 characters across VAL-SET-046, B = 500 RuNIE replicates, Z = +5.58 SD), uniting 248 Dayhoff-6 + CAT-GTR nuclear orthologs, 24-BLG chromosome-scale macrosynteny, 34 miRNA families, Hox signature peptides, and 14 fossil tip calibrations.

Why did virtually all modern animal body plans—from arthropods, mollusks, and brachiopods to echinoderms and chordates—appear so abruptly in the Cambrian fossil record between 539 and 514 million years ago (Ma)? Unfiltered amino-acid molecular clocks push the origin of bilaterian animals deep into the Cryogenian–Tonian (780–830 Ma, implying a 250-million-year "ghost interval" with no fossils), whereas literal fossil reading compresses the entire animal tree below 539 Ma. Here we analyze 1.84 million aligned genomic sites, 24 ancestral chromosome linkage groups (24-BLG), 34 microRNA families, Hox developmental peptides, and 312 anatomical characters across 54 living and 14 iconic Ediacaran–Cambrian fossils (Kimberella, Ikaria, Dickinsonia, Anomalocaris, Hallucigenia, Haikouichthys, Pikaia) using RuNIE across 500 bootstrap replicates. We show that extreme amino-acid compositional bias and a 4.46-fold clock speedup in fast-evolving acoel flatworms and nematodes vs. slow-evolving corals artificially stretch strict clocks by +234.6 million years (to 802.8 Ma). Correcting these biases resolves a two-phase radiation: Crown Bilateria diverged in the Late Ediacaran at 568.2 Ma (Protostomia at 558.4 Ma, Deuterostomia at 554.6 Ma, Spiralia at 546.2 Ma, Ecdysozoa at 542.8 Ma), followed across the Terreneuvian and Cambrian Stage 3 (538.8–514.0 Ma, peaking at 518.4 Ma) by a synchronous 3.86-fold morphological and 2.44-fold molecular rate surge driven by parallel biomineralization (alpha-Carbonic Anhydrase) and a predator–prey visual opsin arms race.

The Mammalian Radiation Supertree: Resolving the Jurassic–Cretaceous Stem and Post-K–Pg Placentalia Crown Chronogram Across 66 Extant and Fossil Taxa (1.72 Mb)

A single-column phylogenomic and paleontological preprint monograph synthesizing 66 extant and Jurassic–Paleocene fossil mammalian and amniote outgroup taxa (1.72 Mb across VAL-SET-045, B = 500 RuNIE replicates, Z = +5.54 SD), uniting 1,240 neutral UCE flanking loci, 318 GC3-masked nuclear coding orthologs, 422 retrotransposon insertions, and 12 fossil tip calibrations.

When did modern placental mammals—from shrews, bats, and rodents to primates, carnivores, elephants, and whales—diversify relative to the 66.02-million-year-old (Ma) Chicxulub asteroid impact that ended the age of non-avian dinosaurs? Unfiltered third-codon molecular clocks push modern orders deep into the Mid-Cretaceous (105–125 Ma), whereas strict fossil tip-dating compresses the entire placental crown after 65 Ma. Here we analyze 1.72 million aligned genomic sites, 1,240 ultraconserved element (UCE) flanking loci, 422 retrotransposon insertions, and 12 Jurassic–Paleocene fossils across 66 taxa using RuNIE across 500 bootstrap replicates. We show that GC-biased gene conversion (gBGC) in third-codon positions (>75% GC3) and a 4.17-fold clock speedup in short-generation rodents vs. long-lived whales artificially stretch strict clocks by +40.2 million years (to 118.4 Ma). Correcting these biases resolves the Soft-Explosive K–Pg Model: Crown Mammalia split at 184.6 Ma, Crown Theria at 158.4 Ma, and the basal Placentalia split between Atlantogenata (Xenarthra + Afrotheria) and Boreoeutheria (Euarchontoglires + Laurasiatheria) occurred in the Late Campanian at 78.2 Ma, followed immediately after the 66.02 Ma K–Pg extinction by an explosive Paleocene burst of all 18 modern placental orders at 64.8 Ma.

Tonian–Cambrian Time-Tree Chronograms Across Ctenophora, Porifera, Placozoa, Cnidaria, and Bilateria: Integrating 29-ALG Chromosome Synteny, Dayhoff-6 LBA Rate Correction, and Ediacaran Fossil Tip-Dating Across 64 Taxa (1.64 Mb)

A single-column phylogenomic and paleontological preprint monograph synthesizing 64 extant and Ediacaran–Cambrian fossil holozoan and metazoan taxa (1.64 Mb across VAL-SET-044, B = 500 RuNIE replicates, Z = +5.48 SD), uniting 7 irreversible Benthozoan BCnS chromosome fusions (p = 1.4 × 10⁻²¹) with Dayhoff-6 + CAT-GTR recoding and LSD2 relaxed clocks.

Which animal group branched off first—sponges (Porifera) or comb jellies (Ctenophora)—and when did the earliest animals diverge relative to the Ediacaran and Cambrian fossil record? Here we analyze 1.64 million aligned genomic sites and 29 ancestral chromosome linkage groups across 64 living and fossil species using RuNIE across 500 bootstrap replicates. We show that pelagic comb jellies evolve 3.42 times faster in nuclear genes and over 12 times faster in mitochondrial DNA than benthic sponges and sea anemones. When analyzed under a strict molecular clock, this rate asymmetry artificially stretches the animal root back to 1,148 million years ago (Ma). By correcting amino-acid compositional bias (reducing RCFV by 83.8%), polarizing the root with 7 irreversible chromosome fusion-with-mixing events shared by sponges, placozoans, cnidarians, and bilaterians, and calibrating an autocorrelated relaxed clock with 10 Ediacaran–Cambrian fossils (†Eoandromeda 551 Ma, †Auroralumina 560 Ma, †Kimberella 555 Ma, †Vauxia 508 Ma, †Haikouichthys 518 Ma), we place Crown Metazoa (the Ctenophora split) in the Late Tonian at 792.4 Ma, the Porifera split at 738.6 Ma, the Placozoa split at 684.2 Ma, and the Cnidaria–Bilateria split at 642.5 Ma.

Reconstructing the Primate Evolutionary Time-Tree (73.4 to 0.0 Ma) Across 56 Extant and Fossil Taxa: K–Pg Radiation, the Hominoid Rate Slowdown, and Pleistocene Ancient-DNA Tip-Dating

A single-column phylogenomic and paleontological preprint monograph synthesizing 56 extant, Pleistocene ancient-DNA, and Paleogene/Neogene fossil primate and euarchontan taxa (1.42 Mb across VAL-SET-041, B = 500 RuNIE replicates, Z = +5.48 SD), reconciling the 2.33-fold hominoid generation-time rate slowdown and excising a human chromosome 5p13.3 nuclear mitochondrial pseudogene (NT_006576.16) via Constrained Least-Squares Dating (LSD2) and autocorrelated log-normal relaxed clocks.

Estimating an accurate geological timeline for primate evolution requires reconciling two persistent sources of systematic bias: the pronounced slowdown in molecular substitution rates accompanying increased body mass and generation time in great apes (Hominoidea), and cryptic nuclear mitochondrial pseudogenes (NUMTs) that distort branch lengths in mitogenomic supermatrices. In this 56-taxon, 1,420,569-site (1.42 Mb) synthesis, we combine Constrained Least-Squares Dating (LSD2), autocorrelated log-normal relaxed molecular clocks, and reading-frame purifying selection filters with 12 heterochronous fossil and Pleistocene ancient-DNA tip calibrations across B = 500 RuNIE bootstrap replicates. The resulting time-tree chronograms place the Crown Primates divergence at 65.80 Ma [95% CI: 64.20–67.40 Ma] across the Cretaceous–Paleogene (K–Pg) boundary, date the trans-Atlantic Simiiformes split to 43.20 Ma [41.50–44.90 Ma], and recover a 6.57 Ma [6.36–6.79 Ma] Pan–Homo divergence after correcting for a 2.33-fold hominoid rate slowdown and excising a chromosome 5p13.3 pseudogene contamination artifact.

From Jurassic Paravian Theropods to the K–Pg Neoaves Radiation: Time-Tree Chronograms (246.5 to 0.0 Ma) Across 62 Dinosaurian and Avian Taxa

A single-column phylogenomic and paleontological preprint monograph synthesizing 62 extant, Holocene ancient-DNA, and Mesozoic/Paleogene fossil archosaurian, theropod, and avian taxa (1.54 Mb across VAL-SET-042, B = 500 RuNIE replicates, Z = +5.62 SD), resolving the Avian "Rocks vs. Clocks" discrepancy via Constrained Least-Squares Dating (LSD2), autocorrelated relaxed clocks, and microchromosomal GC3/NUMT filtering.

For over two decades, avian macroevolution has been divided by a 35-to-45-million-year discrepancy between strict molecular clocks—which place the radiation of modern birds (Neoaves) deep in the Early Cretaceous (100–115 Ma)—and the paleontological record, where crown neoavian fossils appear abruptly after the 66.0 Ma Cretaceous–Paleogene (K–Pg) boundary. Here we synthesize 1,540,000 aligned sites (1.54 Mb) across 62 extant, Holocene ancient-DNA, and Mesozoic/Paleogene fossil archosaurian and avian taxa using RuNIE (Rust Native Inference Engine), Constrained Least-Squares Dating (LSD2), autocorrelated log-normal relaxed clocks, and dual-pass blinded compositional filtering across B = 500 bootstrap replicates. Excising GC3-saturated microchromosomal coding exons (GC3 = 86.4%) and a chromosome 2 nuclear mitochondrial pseudogene (NC_052533.1) while modeling the 2.84-fold post-Chicxulub substitution-rate surge places Paraves at 162.50 Ma, Crown Aves at 89.40 Ma, and Crown Neoaves at 66.80 Ma [95% CI: 65.40–68.20 Ma]—bringing genomic time-trees into direct quantitative concordance with the fossil record.

Out of Africa Through the L3-to-M/N Mitogenome Bottleneck: Time-Tree Chronograms, Haplotype k-Core Hubs, and OXPHOS Purifying Selection Across 58 Hominin Genomes

A single-column paleogenomic preprint monograph synthesizing 58 extant human, ancient Upper Paleolithic Homo sapiens, and archaic hominin complete mitochondrial genomes (1.26 Mb across 32 cohorts, B = 50 RuNIE replicates, Z = +5.34 SD), resolving the 71.8 ka East African L3 root, the 60.4–61.2 ka M/N dispersal bottleneck, Dual-Pass SHA-256 NUMT/aDNA quarantine, and Complex IV OXPHOS 2nd-codon purifying selection.

Every indigenous maternal lineage outside Africa traces back to a single East African mitochondrial ancestor known as haplogroup L3. By analyzing 58 complete modern, ancient Upper Paleolithic, and archaic hominin mitochondrial genomes (1.26 Mb across 32 cohorts) with RuNIE (Rust Native Inference Engine), we show that Dual-Pass SHA-256 blinded auditing automatically filters out nuclear pseudogene (NUMT) and ancient DNA damage artifacts, dating the East African L3 expansion to 71.8 ka and the twin Out-of-Africa founder branches M (60.4 ka) and N (61.2 ka) to a narrow 55–65 ka dispersal window while demonstrating 100% 2nd-codon invariance across the Complex IV oxygen-reduction core.

RuNIE (Rust Native Inference Engine): An Open-Source Architecture for Autonomous Bioinformatics Pipeline Synthesis, Blinded Validation, and Bayesian Scope-Depth Auditing

A technical preprint monograph documenting the 35 native Rust engines of crvscience-labs, the 4-generation parallel-tempering GA-MCMC pipeline synthesizer, Dual-Pass SHA-256 blinded validation, why an orthogonal multi-stage Scope × 0–5 Beaker Bayesian depth system replaces frequentist supermatrix p-values, and a synthesis of 26 Flagged Critical Reports.

Modern bioinformatics workflows often chain separate command-line programs through fragile shell scripts, where hidden formatting mismatches, database misannotations, and model misspecifications can produce 100% bootstrap support and tiny p-values for incorrect evolutionary trees. Here we present RuNIE (Rust Native Inference Engine), an open-source (GNU GPL-3.0) Rust architecture that unites 35 head-to-head validated bioinformatics tools with Dual-Pass SHA-256 blinded auditing and an orthogonal multi-stage Scope × 0–5 Beaker Bayesian calibration system across 42 validation datasets and 26 Flagged Critical Reports.