Winged insects (Pterygota) comprise three principal lineages — mayflies (Ephemeroptera), dragonflies and damselflies (Odonata), and all remaining winged insects (Neoptera) — and which of the three possible branching orders at their common base is correct (the Palaeoptera, Metapterygota, or Chiastomyaria hypothesis) remains one of the classic open questions of insect systematics, with direct consequences for scenarios of the origin of insect wings and flight. A 2010 mitochondrial-genome (mitogenome) study of the damselfly Euphaea formosa exemplified the difficulty: it recovered none of the three classical arrangements, instead grouping Ephemeroptera with Plecoptera (stoneflies) and rendering Neoptera non-monophyletic. The subsequent literature shows: nuclear phylogenomics and site-heterogeneous mitogenomic analyses now support monophyletic Neoptera, and bear on whether the anomalous 2010 grouping is a model artifact rather than biological history; monophyletic Zygentoma (silverfish) including the relic Tricholepidion gertschi is well supported; yet the central trichotomy remains contested, with large studies after 2018 still supporting different hypotheses and an explicit 2018 assessment declaring the problem open. The problem is formulated as: determine the true relationships among Ephemeroptera, Odonata, and Neoptera — that is, decide among the Palaeoptera, Metapterygota and Chiastomyaria hypotheses — and establish whether mitochondrial-genome data, with expanded sampling at the base of Neoptera, can be trusted to answer this deep radiation rather than producing artifacts such as the recovered Ephemeroptera+Plecoptera grouping.

Background

Insects are divided into wingless lineages — bristletails (Archaeognatha) and silverfish (Zygentoma) — and the winged insects or Pterygota. The Pterygota split into the Palaeoptera sensu lato candidates and the Neoptera: neopterans can fold their wings flat over the abdomen, whereas mayflies (Ephemeroptera) and dragonflies/damselflies (Odonata) cannot. Three mutually exclusive hypotheses describe the earliest pterygote split: (1) the Palaeoptera hypothesis — Ephemeroptera + Odonata form a clade sister to Neoptera; (2) the Metapterygota hypothesis — Ephemeroptera branched first, Odonata being sister to Neoptera; (3) the Chiastomyaria (basal Odonata) hypothesis — Odonata branched first, Ephemeroptera being sister to Neoptera. Deciding among them matters because each implies a different scenario for the origin of insect wings and flight. The metazoan mitochondrial genome (mitogenome) is a small circular molecule with 37 genes (13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes), and in the 2000s it was the most readily available genome-scale marker for deep insect phylogeny; intergenic spacers (non-coding DNA between genes; in odonate mitogenomes such spacers have been labelled s1–s5, although the counting and numbering of spacer regions is not consistent between mitogenome publications — Herzog et al. 2016) have been proposed as synapomorphies, i.e. shared derived characters diagnosing a clade. Deep mitogenomic inference is, however, vulnerable to systematic error: strong AT compositional bias, among-lineage rate heterogeneity, and saturation cause long-branch attraction (the artifactual grouping of fast-evolving lineages that sit on long phylogenetic branches), and standard site-homogeneous substitution models (one process for all sites, e.g. the general time-reversible model, GTR) do not correct this, whereas site-heterogeneous models (e.g. CAT-GTR, which let substitution profiles vary across sites) often do. A phylogenomic study based on expressed sequence tags (ESTs — single-pass sequenced cDNA fragments used as gene markers; Simon et al. 2009) had supported Chiastomyaria. Against this background, Lin, Chen and Huang (2010) sequenced the first complete damselfly mitogenome (Euphaea formosa, 15,700 bp) and analyzed 13 mitochondrial protein-coding genes for 25 hexapods; their maximum-parsimony, maximum-likelihood, and Bayesian analyses rejected support for all three classical hypotheses and instead recovered a strongly supported basal Odonata together with an unexpected sister grouping of Ephemeroptera and Plecoptera (stoneflies, a neopteran order), contradicting the near-universally accepted monophyly of Neoptera. They reported the presence of the intergenic spacer s5 as a likely synapomorphy for the dragonflies (Anisoptera), and concluded that the unexpected Ephemeroptera+Plecoptera clade 'requires further analyses with additional mitochondrial genome sampling at the base of the Neoptera'. Related wingless-lineage context: the clade Zygentoma + Pterygota is termed Dicondylia, and silverfish (Zygentoma), including the relict Tricholepidion gertschi, are conventionally used as the wingless outgroup to Pterygota; the interrelationships of Zygentoma and the placement of T. gertschi are a separate, largely morphology-driven question (Blanke et al. 2014) and are not part of the problem posed here.

Problem Statement

Determine the branching order of the three principal lineages of winged insects — mayflies (Ephemeroptera), dragonflies and damselflies (Odonata), and the remaining winged insects (Neoptera) — i.e., decide among the Palaeoptera hypothesis (Ephemeroptera + Odonata sister to Neoptera), the Metapterygota hypothesis (Ephemeroptera first-branching, Odonata sister to Neoptera), and the Chiastomyaria hypothesis (Odonata first-branching, Ephemeroptera sister to Neoptera). As posed by the Euphaea formosa mitogenome study, whose maximum-parsimony, maximum-likelihood and Bayesian analyses 'cannot support the three existing phylogenetic hypotheses of the basal Pterygota (Palaeoptera, Metapterygota, and Chiastomyaria)' and whose conflicting result — the unexpected Ephemeroptera+Plecoptera clade, contradicting a monophyletic Neoptera — 'requires further analyses with additional mitochondrial genome sampling at the base of the Neoptera' (Lin et al. 2010), the problem additionally asks whether mitochondrial-genome data, with expanded taxon sampling at the base of Neoptera, can yield a trustworthy answer to this deep and apparently rapid radiation rather than artifacts like the recovered Ephemeroptera+Plecoptera sister relationship and non-monophyletic Neoptera. These are parts of one objective: a robust reconstruction of the earliest winged-insect radiation that decides among the three named hypotheses and identifies which inference problems are biological history and which are artifacts of data and models. A complete answer weighs the evidence for each of the three hypotheses and names the arrangement best supported by the cited data; where the evidence does not decide among them, the answer must say so explicitly and justify that conclusion. Wingless silverfish (Zygentoma) serve as the outgroup; their own interrelationships, and the position of the relict Tricholepidion gertschi, are a separate question and are not part of this objective.

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