The complete mitochondrial genome of the cockroach Eupolyphaga sinensis (Zhang et al., 2010) left several relationships open: the deeper relationships within Dictyoptera, the basal split among the winged-insect lineages Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), and Neoptera (all other winged insects), and the weakly supported placements of Campodeoidea (Diplura) and Collembola in the hexapod tree. Later literature has decisively confirmed the first component: cockroaches including termites are sister to mantises, with termites nested as eusocial cockroaches. The other two components remain open. Mitogenome-scale and phylogenomic studies continue to recover different basal pterygote topologies — Palaeoptera, Metapterygota, or Chiastomyaria — with support that flips with outgroup choice and substitution model, and the relationships of the primarily wingless hexapods (Protura, Collembola, Diplura) to the true insects remain contested, with the most comprehensive site-heterogeneous phylogenomic analysis (2024) supporting a 'Protura-sister' topology and a monophyletic Collembola + Diplura clade against earlier nuclear-gene and microRNA results, and a published 2025 dispute over that conclusion still unresolved. The open problem is to resolve these basal nodes of the hexapod phylogeny with evidence robust to systematic error and congruent across independent data classes.

Background

Insects are the most species-rich animal group, and the deepest splits in their phylogeny have been debated for over a century. Winged insects (the clade Pterygota) include two basal orders with aquatic juveniles that cannot fold their wings flat over the abdomen — mayflies (Ephemeroptera) and dragonflies and damselflies (Odonata) — and the wing-folding Neoptera (all remaining winged insects, from stoneflies and cockroaches to beetles, moths, and flies). Three mutually exclusive hypotheses compete for the relationships among these three lineages: the Palaeoptera hypothesis ((Ephemeroptera + Odonata) + Neoptera), the Metapterygota hypothesis (Ephemeroptera + (Odonata + Neoptera)), and the Chiastomyaria hypothesis (Odonata + (Ephemeroptera + Neoptera)). Which one is true constrains scenarios for the origin of insect wings and flight — in particular whether the ancestor of winged insects passed through an aquatic juvenile stage. A parallel century-old controversy concerns the 'primarily wingless' hexapods: springtails (Collembola), proturans (Protura), and two-pronged bristletails (Diplura; Campodeoidea is a dipluran superfamily), often grouped as Entognatha for their internalized mouthparts, versus the true insects (Ectognatha: bristletails, silverfish, and all winged insects). Competing topologies here — monophyletic Entognatha, Ellipura (Protura + Collembola), Nonoculata (Protura + Diplura), or Diplura as the closest relative of Ectognatha — imply different scenarios for the evolution of the insect body plan and for the terrestrialization of six-legged animals (Hexapoda). A complete mitochondrial genome (mitogenome) is the small circular molecule, about 15–16 thousand base pairs, encoding 13 respiratory-chain proteins plus rRNAs and tRNAs; because mitogenomes became cheap to sequence for many species, they were the workhorse data for these deep divergences, but they evolve fast and their base-composition biases are a known source of systematic error (attractive grouping of unrelated fast-evolving lineages, so-called long-branch attraction, and sensitivity to the choice of outgroups used to root the tree). Against this background, Zhang et al. (2010) sequenced the mitogenome of the cockroach Eupolyphaga sinensis and, analyzing the concatenated mitochondrial proteins of 24 arthropods, recovered the cockroaches (Blattaria) including the termites (Isoptera) as sister to the mantises (Mantodea) — together the Dictyoptera — while the basal pterygote resolution matched none of the three classical hypotheses: the sampled mayfly Parafronurus youi clustered instead with the stonefly Pteronarcys princes (Plecoptera, a neopteran), and the deeper nodes, including the placements of Campodeoidea and Collembola, were only weakly supported. The authors therefore flagged the deeper dictyopteran relationships, the basal split of the winged insects, and the entognathan placements as unresolved pending broader sampling. Subsequent nuclear phylogenomics (genome-scale analysis of hundreds to over a thousand genes) resolved many previously contested insect relationships (Misof et al. 2014) and confirmed the dictyopteran result, but the basal pterygote and entognathan nodes have remained refractory, with different data classes and substitution models supporting different topologies.

Problem Statement

Resolve the two still-open basal nodes of the hexapod phylogeny raised by the Eupolyphaga sinensis mitogenome study, whose dictyopteran component has since been settled: (1) Determine the true relationships among the three basal lineages of winged insects — Ephemeroptera (mayflies), Odonata (dragonflies and damselflies), and Neoptera — that is, decide among the Palaeoptera hypothesis ((Ephemeroptera + Odonata) + Neoptera), the Metapterygota hypothesis (Ephemeroptera + (Odonata + Neoptera)), and the Chiastomyaria hypothesis (Odonata + (Ephemeroptera + Neoptera)), and explain why the competing mitochondrial-genome and phylogenomic results that currently support different topologies disagree (outgroup choice, substitution model, composition heterogeneity, long-branch attraction). (2) Determine the phylogenetic positions of the early-diverging hexapod lineages — Diplura (including Campodeoidea), Collembola, and Protura — relative to Ectognatha (true insects), that is, decide among the competing topologies (monophyletic Entognatha (Collembola + Diplura + Protura), Ellipura (Protura + Collembola), Nonoculata (Protura + Diplura), Diplura sister to Ectognatha, or Protura sister to all other hexapods). A complete answer establishes, for each node, a topology whose support is robust to outgroup choice and model specification (including site-heterogeneous models) and is congruent across independent data classes such as nuclear phylogenomics, mitochondrial genomes, morphology, and rare genomic changes; an answer resolving only one of the two nodes is a partial answer.

References

  1. Zhang YY, Xuan WJ, Zhao JL, Zhu CD, Jiang GF (2010) The complete mitochondrial genome of the cockroach Eupolyphaga sinensis (Blattaria: Polyphagidae) and the phylogenetic relationships within the Dictyoptera. Molecular Biology Reports 37(7):3509-3516. DOI: 10.1007/s11033-009-9944-1. https://doi.org/10.1007/s11033-009-9944-1
  2. Inward D, Beccaloni G, Eggleton P (2007) Death of an order: a comprehensive molecular phylogenetic study confirms that termites are eusocial cockroaches. Biology Letters 3(3):331-335. DOI: 10.1098/rsbl.2007.0102. https://doi.org/10.1098/rsbl.2007.0102
  3. Legendre F, Nel A, Svenson GJ, Robillard T, Pellens R, Grandcolas P (2015) Phylogeny of Dictyoptera: Dating the origin of cockroaches, praying mantises and termites with molecular data and controlled fossil evidence. PLoS ONE 10(7):e0130127. DOI: 10.1371/journal.pone.0130127. https://doi.org/10.1371/journal.pone.0130127
  4. Evangelista DA, Wipfler B, Bethoux O, Donath A, Fujita M, Kohli MK, Legendre F, Liu S, Machida R, Misof B, Peters RS, Podsiadlowski L, Rust J, Schuette K, Tollenaar W, Ware JL, Wappler T, Zhou X, Meusemann K, Simon S (2019) An integrative phylogenomic approach illuminates the evolutionary history of cockroaches and termites (Blattodea). Proceedings of the Royal Society B 286(1895):20182076. DOI: 10.1098/rspb.2018.2076. https://doi.org/10.1098/rspb.2018.2076
  5. Wipfler B, Letsch H, Frandsen PB, Kapli P, Mayer C, Bartel D, Buckley TR, Donath A, Edgerly-Rooks JS, Fujita M, Liu S, Machida R, Mashimo Y, Misof B, Niehuis O, Peters RS, Petersen M, Podsiadlowski L, Schutte K, Shimizu S, Uchifune T, Wilbrandt J, Yan E, Zhou X, Simon S (2019) Evolutionary history of Polyneoptera and its implications for our understanding of early winged insects. Proceedings of the National Academy of Sciences USA 116(8):3024-3029. DOI: 10.1073/pnas.1817794116. https://doi.org/10.1073/pnas.1817794116
  6. Misof B, Liu S, Meusemann K, Peters RS, Donath A, Mayer C, Frandsen PB, Ware J, Flouri T, Beutel RG, Niehuis O, Petersen M, Izquierdo-Carrasco F, Wappler T, Rust J, Aberer AJ, Aspoeck U, Aspoeck H, Bartel D, Blanke A, Berger S, Boehm A, Buckley TR, Calcott B, Chen J, Friedrich F, Fukui M, Fujita M, Greve C, Grobe P, Gu S, Huang Y, Jermiin LS, Kawahara AY, Krogmann L, Kubiak M, Lanfear R, Letsch H, Li Y, Li Z, Li J, Lu H, Machida R, Mashimo Y, Kapli P, McKenna DD, Meng G, Nakagaki Y, Navarrete-Heredia JL, Ott M, Ou Y, Pass G, Podsiadlowski L, Pohl H, von Reumont BM, Schutte K, Sekiya K, Shimizu S, Slipinski A, Stamatakis A, Song W, Su X, Szucsich NU, Tan M, Tan X, Tang M, Tang J, Timelthaler G, Tomizuka S, Trautwein M, Tong X, Uchifune T, Walzl MG, Wiegmann BM, Wilbrandt J, Wipfler B, Wong TK, Wu Q, Wu G, Xie Y, Yang S, Yang Q, Yeates DK, Yoshizawa K, Zhang Q, Zhang R, Zhang W, Zhang Y, Zhao J, Zhou C, Zhou L, Ziesmann T, Zou S, Li Y, Xu X, Zhang Y, Yang H, Wang J, Wang J, Kjer KM, Zhou X (2014) Phylogenomics resolves the timing and pattern of insect evolution. Science 346(6210):763-767. DOI: 10.1126/science.1257570. https://doi.org/10.1126/science.1257570
  7. Sasaki G, Ishiwata K, Machida R, Miyata T, Su ZH (2013) Molecular phylogenetic analyses support the monophyly of Hexapoda and suggest the paraphyly of Entognatha. BMC Evolutionary Biology 13:236. DOI: 10.1186/1471-2148-13-236. https://doi.org/10.1186/1471-2148-13-236
  8. Dell'Ampio E, Meusemann K, Szucsich NU, Peters RS, Meyer B, Borner J, Petersen M, Aberer AJ, Stamatakis A, Walzl MG, Minh BQ, von Haeseler A, Ebersberger I, Pass G, Misof B (2014) Decisive data sets in phylogenomics: lessons from studies on the phylogenetic relationships of primarily wingless insects. Molecular Biology and Evolution 31(1):239-249. DOI: 10.1093/molbev/mst196. https://doi.org/10.1093/molbev/mst196
  9. Simon S, Blanke A, Meusemann K (2018) Reanalyzing the Palaeoptera problem - The origin of insect flight remains obscure. Arthropod Structure and Development 47(4):328-338. DOI: 10.1016/j.asd.2018.05.002. https://doi.org/10.1016/j.asd.2018.05.002
  10. Song N, Li X, Yin X, Li X, Yin J, Pan P (2019) The mitochondrial genomes of palaeopteran insects and insights into the early insect relationships. Scientific Reports 9:17765. DOI: 10.1038/s41598-019-54391-9. https://doi.org/10.1038/s41598-019-54391-9
  11. Yu DN, Yu PP, Zhang LP, Storey KB, Gao XY, Zhang JY (2021) Increasing 28 mitogenomes of Ephemeroptera, Odonata and Plecoptera support the Chiastomyaria hypothesis with three different outgroup combinations. PeerJ 9:e11402. DOI: 10.7717/peerj.11402. https://doi.org/10.7717/peerj.11402
  12. Liu A, Chen W, Huang C, Qian C, Liang Y, Li S, Zhan S, Luan Y (2020) MicroRNA evolution provides new evidence for a close relationship of Diplura to Insecta. Systematic Entomology 45(2):365-377. DOI: 10.1111/syen.12401. https://doi.org/10.1111/syen.12401
  13. Du S, Tihelka E, Yu D, Chen WJ, Bu Y, Cai C, Engel MS, Luan YX, Zhang F (2024) Revisiting the four Hexapoda classes: Protura as the sister group to all other hexapods. Proceedings of the National Academy of Sciences USA 121(39):e2408775121. DOI: 10.1073/pnas.2408775121. https://doi.org/10.1073/pnas.2408775121
  14. Machida R, Fukui M, Tomizuka S, Ikeda Y, Sekiya K, Masumoto M (2025) Embryology cannot establish the "Protura-sister". Proceedings of the National Academy of Sciences USA 122(5):e2423813122. DOI: 10.1073/pnas.2423813122. https://doi.org/10.1073/pnas.2423813122
  15. Du S, Tihelka E, Yu D, Chen WJ, Bu Y, Cai C, Engel MS, Luan YX, Zhang F (2025) Reply to Machida et al.: Although embryology cannot establish the "Protura-sister", phylogenomics can. Proceedings of the National Academy of Sciences USA 122(5):e2425775122. DOI: 10.1073/pnas.2425775122. https://doi.org/10.1073/pnas.2425775122
  16. Almudi I, Vizueta J, Wyatt CDR, de Mendoza A, Marletaz F, Firbas PN, Feuda R, Masiero G, Medina P, Alcaina-Caro A, Cruz F, Gomez-Garrido J, Gut M, Alioto TS, Vargas-Chavez C, Davie K, Misof B, Gonzalez J, Aerts S, Lister R, Paps J, Rozas J, Sanchez-Gracia A, Irimia M, Maeso I, Casares F (2020) Genomic adaptations to aquatic and aerial life in mayflies and the origin of insect wings. Nature Communications 11:2632. DOI: 10.1038/s41467-020-16284-8. https://doi.org/10.1038/s41467-020-16284-8