The phylogenomic study of Misof et al. (Science, 2014) inferred the insect tree from 1,478 protein-coding genes and dated the origin of insects to the Early Ordovician (~479 million years ago, Ma). Three issues arising from that study remain unresolved in the literature that followed: the relationships among the earliest winged insect lineages (mayflies, dragonflies and damselflies, and all other winged insects), inferred there with sparse palaeopteran taxon sampling; the placement of Psocodea (bark lice and parasitic lice) as sister group of the holometabolous insects, which conflicted with the traditional Paraneoptera and was not favored by the study's own four-cluster likelihood mapping and permutation tests; and the reconciliation of the molecular dates with the complete absence of unequivocal Cambrian–Silurian insect fossils. Later work through 2026 has corroborated but not settled any of the three: phylogenomic and mitogenomic analyses still disagree on the basal pterygote split, the largest dedicated analysis still finds the Psocodea–Holometabola grouping supported by tree inference but not by likelihood-mapping tests, and the pre-Devonian fossil gap persists while its interpretation remains debated. The problem is to resolve these three separately open questions about the deep insect tree and its early timescale: the basal pterygote relationships, the true sister group of Holometabola, and the reconciliation of the inferred Ordovician origins of insects with the fossil record through a documented re-diagnosis of candidate pre-Devonian insect or stem-hexapod fossils and/or integrative recalibration.

Background

Insects (class Insecta) account for the majority of described animal species, so the branching order of their major lineages is one of the most consequential questions in evolutionary biology. Winged insects (Pterygota) are divided into Palaeoptera — mayflies (Ephemeroptera) and dragonflies and damselflies (Odonata), which cannot fold their wings flat over the abdomen — and Neoptera, all other winged insects, which can. Because both living palaeopteran orders retain many ancestral traits and their Mesozoic-to-Paleozoic stem fossils are mostly known from wings, three competing hypotheses for the earliest splits among winged insects have coexisted for decades: Palaeoptera monophyly (Ephemeroptera + Odonata as a clade, sister to Neoptera); Metapterygota (Ephemeroptera branches first, Odonata is sister to Neoptera); and Chiastomyaria (Odonata branches first, Ephemeroptera is sister to Neoptera). Morphological character systems, ribosomal RNA, mitochondrial genomes, and nuclear phylogenomic matrices have each favored different answers, and proposed morphological synapomorphies of Ephemeroptera + Odonata have repeatedly been criticized as plesiomorphic or convergent (this history is summarized in Song et al. 2019).

A second contested deep node concerns Paraneoptera, the 'hemipteroid' assemblage comprising Psocodea (bark lice and parasitic lice), Thysanoptera (thrips), and Hemiptera (true bugs and allies), versus Holometabola, the clade of insects with complete metamorphosis (beetles, wasps, flies, butterflies and others) that contains more than half of all insect species. Morphological evidence supports a monophyletic Paraneoptera, yet several molecular analyses have instead placed Psocodea as the sister group of Holometabola, which would render Paraneoptera paraphyletic.

Phylogenomics — the inference of evolutionary trees from genome-scale data such as the 1,478 protein-coding genes assembled by Misof et al. (2014) from transcriptomes (the set of RNA transcripts of expressed genes) of taxa across all insect orders — resolved much of the ordinal-level backbone. Notably, it supported Palaeoptera monophyly (with sparse palaeopteran taxon sampling) and recovered Psocodea as sister to Holometabola. However, the authors reported that this grouping did not hold under all statistical tests: in particular, four-cluster likelihood mapping (FcLM), a technique that scores how strongly a dataset favors each of the possible resolutions of a four-taxon split around an internal node, together with permutation tests, attributed much of the likelihood for the Psocodea + Holometabola clade to noise in the data (as described by Wang et al. 2016 and Johnson et al. 2018).

The third thread concerns timing. Using relaxed-clock molecular dating — models in which substitution rates vary across branches, calibrated by fossils — Misof et al. dated the origin of insects to the Early Ordovician (~479 Ma) and the origin of insect flight to the Early Devonian (~406 Ma). Yet the oldest widely accepted insect fossils are Early Devonian in age (roughly 400–411 Ma, for example the mandibles of Rhyniognatha hirsti from the Rhynie chert of Scotland), and the insect fossil record only becomes rich from the Carboniferous. A stem group is the set of extinct taxa closer to a living clade than to any other living clade but outside its crown group (the last common ancestor of the living members and all of its descendants); no unequivocal Cambrian, Ordovician, or Silurian insect or insect stem-group fossil is known, so the earliest ~70 million years of inferred insect history have no direct fossil record. Whether this gap reflects a genuine cryptic early history (possibly in terrestrial or even aquatic habitats that preserve poorly), overestimated molecular dates due to calibration and rate-model artifacts, or simply unfound fossils, remains actively debated (Tihelka et al. 2022); a reanalysis of the same data argued in the opposite direction, that the dating method systematically underestimated the timescale and that key insect groups evolved up to 100 million years earlier than inferred there (Tong et al. 2015). Closely related open questions in the same literature are the timing of the origin of insect flight — reported as ~406 Ma by Misof et al., but still treated as contentious by later work (Song et al. 2019; Simon et al. 2018) — and the habitat of the earliest hexapod lineages, which Wang et al. (2016) argue were most likely marine, the transition to land remaining of unknown timing. For comparison, the analogous gap for myriapods (millipedes and centipedes) was narrowed by the discovery of aquatic stem-group fossils (Edgecombe et al. 2020), illustrating what closure of the insect gap would require.

Problem Statement

Resolve the deep nodes and early timescale of insect evolution that remain contested after Misof et al. (2014), by answering the following three linked questions.

  1. Basal pterygote relationships. Determine which of the three competing hypotheses correctly describes the earliest splits among winged insects: Palaeoptera monophyly (Ephemeroptera + Odonata as a clade, sister to Neoptera), Metapterygota (Ephemeroptera as the first-diverging winged insect lineage, Odonata sister to Neoptera), or Chiastomyaria (Odonata first-diverging, Ephemeroptera sister to Neoptera). Support for this node has proven sensitive to model choice (including site-heterogeneous substitution models), outgroup choice and data type: Song et al. (2019) record that the genome-scale analysis of Misof et al. supported Palaeoptera and its sister relationship to Neoptera 'though with limited taxonomic sampling for palaeopteran insects', Yu et al. (2021) instead recover Chiastomyaria under three different outgroup combinations, and Simon, Blanke and Meusemann (2018) treat the Palaeoptera problem as unresolved.

  2. The placement of Psocodea. Determine whether Psocodea is the sister group of Holometabola — making the traditional Paraneoptera (Psocodea + Condylognatha, i.e., Thysanoptera + Hemiptera) paraphyletic — or whether Paraneoptera is monophyletic with Psocodea sister to the remaining paraneopterans. Statistical tests and data types (protein-coding nuclear genes, mitochondrial genomes, morphology) have not agreed on this node: the grouping recovered by Misof et al. was not favored by their own four-cluster likelihood mapping and permutation tests (Wang et al. 2016), and the largest dedicated analysis reproduced the same conflict between maximum-likelihood support and likelihood mapping, concluding that the position of Psocodea 'is still an open question' that requires further study of morphological and molecular data (Johnson et al. 2018).

  3. Reconciliation of the early timescale with the fossil record. Reconcile the Early Ordovician molecular estimate of the origin of insects (~479 Ma in Misof et al.; independent reanalyses give a comparable Ordovician age for the split of stem-group insects from entognathous hexapods, ~475 Ma in Wang et al. 2016, whereas Misof et al. dated the origin of Hexapoda as a whole, a different node, to ~493 Ma) with the absence of unequivocal Cambrian, Ordovician, and Silurian insect or insect stem-group fossils — through a documented re-diagnosis or reinterpretation of candidate pre-Devonian insect or stem-hexapod fossils (whether terrestrial or aquatic), and/or integrative recalibration (total-evidence or fossilized-birth-death dating, refined clock and rate models) — including the habitat in which the earliest hexapod stem lineages lived, since it determines the depositional settings in which such fossils would have to be sought (Wang et al. 2016). A defensible resolution reached by either route — the fossil re-diagnosis or the integrative recalibration — answers this question; the two are alternatives, not joint requirements.

The three questions are linked but separately open: the topologies of items 1 and 2 are the scaffold on which the chronogram of item 3 is estimated and calibrated, and all three concern the same study and the same dataset.

References

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