The first branching events within the winged insects (Pterygota) — whether mayflies (Ephemeroptera) and dragonflies/damselflies (Odonata) form a clade (Palaeoptera hypothesis), whether mayflies are the earliest-diverging winged insects with dragonflies allied to all other winged insects (Metapterygota hypothesis), or whether mayflies group with the remaining winged insects (Chiastomyaria hypothesis) — remain unsettled despite morphological, mitochondrial-genome, and phylogenomic analyses. Different large datasets support each of the three topologies, and demonstrated sensitivity to taxon composition, outgroup choice, and substitution model means no single current matrix yields a trustworthy conclusion. The problem is to determine the basal pterygote branching order with evidence whose outcome is demonstrably robust to these perturbations, thereby deciding whether any single topology — including 'mayflies first' — actually holds.

Background

Winged insects (Pterygota) constitute the overwhelmingly dominant radiation of animals, and their three surviving basal lineages are mayflies (Ephemeroptera), dragonflies and damselflies (Odonata), and Neoptera (all remaining winged insects, which can fold their wings flat over the abdomen, unlike the two palaeopteran orders). Which of these lineages diverged first is a century-old question, the 'Palaeoptera problem', because the answer determines what the common ancestor of all winged insects looked like and in what order the key adaptations of insect flight were acquired. Three mutually exclusive hypotheses compete: (1) the Palaeoptera hypothesis, in which Ephemeroptera + Odonata form a clade (supported morphologically by the inability to fold the wings back, bristle-like antennae, and intercalary wing veins); (2) the Metapterygota hypothesis, in which Odonata + Neoptera form a clade and Ephemeroptera is the sister group of all other winged insects — the 'mayflies diverged first' scenario (supported by the mayfly subimago stage, a unique winged pre-adult molt, and mandibular articulation characters); and (3) the Chiastomyaria hypothesis, in which Ephemeroptera + Neoptera form a clade and Odonata branches off first (supported historically by reproductive characters such as indirect sperm transfer). In phylogenetic inference, an outgroup is a taxon outside the group under study that roots the tree, and its choice can systematically distort deep relationships; taxon sampling is the set of species included in the data matrix. A mitogenome is the mitochondrial genome, a small maternally inherited marker that evolves fast and is prone to compositional bias at deep divergences; phylogenomics instead uses hundreds to thousands of nuclear protein-coding genes, typically from transcriptomes (the set of expressed genes). Site-heterogeneous substitution models (such as CAT-GTR) allow different sites to follow different replacement patterns and are less prone to long-branch attraction — the systematic artifact whereby fast-evolving lineages are erroneously grouped. The empirical state of the field is discordant. Thomas et al. (2013) showed that outgroup choice and limited taxon sampling are major sources of systematic error even in phylogenomic datasets: their taxon-rich 113-taxon analysis consistently supported Palaeoptera, while smaller datasets flip among all three hypotheses under minor changes of data or method. Misof et al. (2014), the largest phylogenomic study (1,478 genes, 144 taxa), recovered monophyletic Palaeoptera, but with few palaeopteran representatives. A dedicated reanalysis by Simon, Blanke and Meusemann (2018) found overall phylogenomic support for Palaeoptera but considerable hidden signal for Chiastomyaria that standard tree inference does not detect, and concluded that the Palaeoptera problem 'has to be considered an open question in insect systematics'. Mitogenome studies disagree with one another: Song et al. (2019), using a site-heterogeneous CAT-GTR model, strongly supported Palaeoptera, whereas Yu et al. (2021), sequencing 28 new mitogenomes and varying outgroup breadth, found Bayesian support for Chiastomyaria and stressed that outgroup choice critically changes the result; the largest odonate mitogenome study to date (Huang et al. 2025) concludes that mitogenomes fall short for higher-level relationships (suborder, superfamily, and interfamily splits), underscoring the limits of this marker class at deep nodes. Even genome-era papers still frame the node differently (Almudi et al. 2020 treat mayflies as sister to all other winged insects). No analysis to date has demonstrated a topology whose recovery is stable under combined perturbations of taxon composition, outgroup breadth, and model class with adequate systematic-error diagnostics.

Problem Statement

Determine the branching order of the three basal pterygote lineages — Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), and Neoptera — i.e., decide which of the three competing hypotheses is correct: Palaeoptera (Ephemeroptera + Odonata as a clade sister to Neoptera), Metapterygota (Odonata + Neoptera as a clade, so that Ephemeroptera is the earliest-diverging extant winged-insect lineage, 'mayflies first'), or Chiastomyaria (Ephemeroptera + Neoptera as a clade, Odonata branching off first) — or conclude, if the robustness evidence so indicates, that none of the three is robustly supported and the basal node is not resolvable. The determination must rest on evidence whose conclusion is robust to the perturbations shown to confound previous studies — taxon composition and sampling (with adequate representation of Ephemeroptera and Odonata), outgroup breadth, substitution-model class (site-homogeneous and site-heterogeneous), and data class (nuclear phylogenomic and/or other) — together with diagnostics for systematic error (compositional heterogeneity, long-branch attraction, within-dataset conflicting signal), demonstrating that the recovered basal relationship is stable across these regimes. A hypothesis is established exactly when it is recovered as the optimal topology under every combination of a pre-declared set of analyses that spans all four perturbation axes above — more than one taxon composition and outgroup configuration (each with adequate representation of Ephemeroptera and Odonata), both substitution-model classes, and the data class or classes named above — and the recovery survives each named diagnostic; this standard is sufficient as well as necessary, so that if no hypothesis meets it, the task's verdict is that the basal node is not resolvable on the evidence examined. The required robustness demonstration may draw on reanalysis of published data matrices, newly assembled data, or both, provided the analysis set actually spans the axes as specified. Yu et al. (2021) frame the open question in terms of sampling and outgroup design: whether increasing taxon sampling and choosing appropriate outgroups can give enough information to obtain an accurate phylogeny of the mayfly/dragonfly/neopteran divergence.

References

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