Pancrustacea — the clade uniting crustaceans with hexapods and containing over 80% of described animal species — still lacks a stable backbone phylogeny at several deep nodes despite genome-scale data. Bernot et al. (2023) showed these nodes are exquisitely sensitive to taxon sampling and analytical method: the position of Copepoda within Allotriocarida differs among maximum-likelihood, site-heterogeneous Bayesian, and coalescent analyses; Ostracoda is non-monophyletic under current sparse ostracod sampling; the placement of Stomatopoda within Malacostraca conflicts between methods; and divergence-time estimates for the deepest nodes swing between Ediacaran and Cambrian ages depending on the relaxed-clock analysis. The peracarid backbone sub-question has been substantially resolved, but the other nodes remain open — most directly confirmed by Yu et al. (2024), who found limited support for any particular relationship among Branchiopoda, Cephalocarida, and Copepoda. The problem is to resolve the surviving nodes — through expanded sampling of the rare taxa that break the longest branches and/or data types beyond single-copy orthologs — such that each resolution is robust across analytical methods, and to identify the source of the clock-model-dependent dating discordance.

Background

Pancrustacea (also called Tetraconata) is the animal clade uniting crustaceans with hexapods — insects plus the wingless Collembola (springtails), Protura (coneheads) and Diplura (two-pronged bristletails). It contains more than 80% of described animal species and about half of animal biomass. Phylogenomic analysis of hundreds of genes has settled that hexapods evolved from within the crustacean tree, yet the branching order of the deep crustacean lineages has remained strikingly unstable across studies — so unstable that several long-recognized higher taxa have been dismantled and reassembled over the past decade.

The deep clades involved are: Oligostraca — ostracods (bivalved seed shrimp), mystacocarids (minute interstitial crustaceans), and ichthyostracans (fish lice and tongue worms) — usually recovered as sister to all other pancrustaceans; and its complement Altocrustacea, which divides into Multicrustacea (Thecostraca, i.e. barnacles and allies; Copepoda, the ubiquitous planktonic and parasitic copepods; and Malacostraca) and Allotriocarida (Branchiopoda, i.e. fairy shrimps, water fleas and allied mostly freshwater forms; Cephalocarida, a rare morphologically primitive interstitial order; Remipedia, venomous cave-dwelling crustaceans; and Hexapoda). These interrelationships bear directly on the origin of insects: depending on the topology, the closest crustacean relatives of hexapods are remipedes, branchiopods, or a grouping involving cephalocarids or even copepods, implying very different scenarios for the evolution of the insect body plan and the timing of terrestrialization.

Three analysis classes dominate the field, and much of the literature's conflict is method conflict: (i) concatenated-supermatrix maximum likelihood (ML) with bootstrap support; (ii) site-heterogeneous Bayesian analysis under the CAT-GTR model in PhyloBayes, in which amino-acid exchange preferences vary across sites — a mitigation for long-branch attraction (LBA), the artefactual grouping of independently fast-evolving lineages — often combined with Dayhoff6 recoding, which collapses amino acids into six categories to damp compositional heterogeneity; and (iii) coalescent summaries of gene trees (e.g. ASTRAL), which model incomplete lineage sorting (ILS), the persistence of ancestral polymorphisms across successive ancient speciations that makes individual gene trees disagree with the species tree.

The prior-results lineage: Regier et al. (2010), in the first arthropod-wide phylogenomic analysis of nuclear protein-coding genes, placed Remipedia + Cephalocarida (the Xenocarida pairing) as sister to Hexapoda. Von Reumont et al. (2012) recovered Remipedia alone as the possible sister group of Hexapoda. Oakley et al. (2013), with then-dense ostracod EST sampling, found a monophyletic Ostracoda. Schwentner et al. (2017) showed that support for competing hexapod sister-group candidates was strongly methodology-dependent and argued the Xenocarida pairing was an LBA artefact. Lozano-Fernandez et al. (2019), with expanded remipede sampling, recovered Allotriocarida with Remipedia sister to Hexapoda and Branchiopoda sister to that pair, and moderate support for Copepoda + Thecostraca in their main analyses. Bernot et al. (2023) analyzed a 105-taxon data set (90 transcriptomes and 15 genomes) with a tree-based orthology pipeline, demonstrated that small changes in taxon sampling flip deep topologies (through effects on the reconstruction method rather than on orthology identification), rejected the classical clades Caridoida, Eucarida, Multicrustacea, Vericrustacea, and Syncarida, and proposed Syneucarida (decapods + euphausiids + syncarids) with Stomatopoda (mantis shrimp) as its sister — together Stomatocarida — dividing Malacostraca into Leptostraca, Stomatocarida, and Peracarida (the brooding marsupial crustaceans: amphipods, isopods, tanaids, cumaceans, mysids and several rare orders; the isopod + tanaid + cumacean clade is termed Mancoida). Most consequentially, all their analyses placed Copepoda inside Allotriocarida with high support — but its position within Allotriocarida differed by method, and Ostracoda came out non-monophyletic under their sparse ostracod sampling. The same paper dated the tree with 13 vetted fossils: an MCMCTree analysis under an independent-rates relaxed clock, using the full amino-acid matrix, placed the deepest pancrustacean divergences in the Ediacaran (roughly 635-539 million years ago), while PhyloBayes analyses, restricted by runtime limits to a 50-locus subsample and run under both autocorrelated and uncorrelated clocks, placed them in the Cambrian (roughly 539-485 million years ago); the cause of the discrepancy was left open, and in the published comparison the clock model is confounded with the locus set. The authors identified 15 rare taxa — among them Platycopioida (the copepod order sister to all other copepods), the early-diverging ostracods Platycopa and Manawa staceyi, and the relict peracarid orders — whose sequencing would break the longest branches, and noted that the set of single-copy orthologs shared across Pancrustacea appears near saturation, so further progress may require paralog-aware or chromosomal-synteny-based methods, which depend on chromosome-scale genome assemblies that remain scarce for non-insect pancrustaceans.

Problem Statement

Determine the resolved, method-robust branching order of the deep pancrustacean backbone at the nodes that remain unstable despite genome-scale data. All four parts below are required, and resolving any single node alone does not complete the task: (1) the interrelationships within Allotriocarida, including the exact position of Copepoda relative to Branchiopoda, Cephalocarida, Remipedia and Hexapoda (maximum-likelihood analyses place Copepoda sister to Branchiopoda, site-heterogeneous CAT-GTR Bayesian analyses place Copepoda + Remipedia sister to Hexapoda, and coalescent ASTRAL analyses place Copepoda sister to all other allotriocaridans); (2) whether Ostracoda is monophyletic, which requires genome-scale sampling of the early-diverging ostracod lineages (the platycopids, including Manawa staceyi) together with additional mystacocarids and branchiurans; (3) the position of Stomatopoda within Malacostraca — sister to Syneucarida (Stomatocarida), as recovered by all ML and coalescent analyses, or in the more basal position recovered by the non-converged CAT-GTR chains; and (4) the source of the discrepancy between the Ediacaran age estimates for the deepest pancrustacean divergences obtained from the MCMCTree analysis of the full amino-acid matrix and the Cambrian ages obtained from the PhyloBayes analyses of a 50-locus subsample (run under both autocorrelated and uncorrelated clocks), disentangling the effect of the clock model from that of locus subsampling and calibration choice. Because the instability at these nodes has manifested primarily as conflict among analytical methods, a resolution of each node should be robust across analysis classes — maximum-likelihood, site-heterogeneous models (Bayesian CAT-GTR or site-heterogeneous ML mixture models such as LG+C60+F+G), and coalescent analyses agreeing on the same topology with strong support at each named node. Resolving these nodes may require expanded sampling of the rare long-branch-breaking taxa (notably Platycopioida copepods, platycopid ostracods, and the remaining cephalocarid genera) and data types beyond single-copy orthologs (paralog-aware and chromosomal-synteny-based inference, contingent on chromosome-scale genome assemblies).

References

  1. Bernot JP, Owen CL, Wolfe JM, Meland K, Olesen J, Crandall KA. 2023. Major Revisions in Pancrustacean Phylogeny and Evidence of Sensitivity to Taxon Sampling. Molecular Biology and Evolution 40(8):msad175. DOI 10.1093/molbev/msad175. https://doi.org/10.1093/molbev/msad175
  2. Bernot JP, Owen CL, Wolfe JM, Meland K, Olesen J, Crandall KA. 2022. Major revisions in pancrustacean phylogeny with recommendations for resolving challenging nodes. bioRxiv preprint. DOI 10.1101/2022.11.17.514186. https://doi.org/10.1101/2022.11.17.514186
  3. Regier JC, Shultz JW, Zwick A, Hussey A, Ball B, Wetzer R, Martin JW, Cunningham CW. 2010. Arthropod relationships revealed by phylogenomic analysis of nuclear protein-coding sequences. Nature 463(7284):1079-1083. DOI 10.1038/nature08742. https://doi.org/10.1038/nature08742
  4. von Reumont BM, Jenner RA, Wills MA, Dell'Ampio E, Pass G, Ebersberger I, Meyer B, Koenemann S, Iliffe TM, Stamatakis A, Niehuis O, Meusemann K, Misof B. 2012. Pancrustacean Phylogeny in the Light of New Phylogenomic Data: Support for Remipedia as the Possible Sister Group of Hexapoda. Molecular Biology and Evolution 29(3):1031-1045. DOI 10.1093/molbev/msr270. https://doi.org/10.1093/molbev/msr270
  5. Oakley TH, Wolfe JM, Lindgren AR, Zaharoff AK. 2013. Phylotranscriptomics to Bring the Understudied into the Fold: Monophyletic Ostracoda, Fossil Placement, and Pancrustacean Phylogeny. Molecular Biology and Evolution 30(1):215-233. DOI 10.1093/molbev/mss216. https://doi.org/10.1093/molbev/mss216
  6. Schwentner M, Combosch DJ, Nelson JP, Giribet G. 2017. A Phylogenomic Solution to the Origin of Insects by Resolving Crustacean-Hexapod Relationships. Current Biology 27(12):1818-1824.e5. DOI 10.1016/j.cub.2017.05.040. https://doi.org/10.1016/j.cub.2017.05.040
  7. Schwentner M, Richter S, Rogers DC, Giribet G. 2018. Tetraconatan phylogeny with special focus on Malacostraca and Branchiopoda: highlighting the strength of taxon-specific matrices in phylogenomics. Proceedings of the Royal Society B 285(1885):20181524. DOI 10.1098/rspb.2018.1524. https://doi.org/10.1098/rspb.2018.1524
  8. Lozano-Fernandez J, Giacomelli M, Fleming JF, Chen A, Vinther J, Thomsen PF, Glenner H, Palero F, Legg DA, Iliffe TM, Pisani D, Olesen J. 2019. Pancrustacean Evolution Illuminated by Taxon-Rich Genomic-Scale Data Sets with an Expanded Remipede Sampling. Genome Biology and Evolution 11(8):2055-2070. DOI 10.1093/gbe/evz097. https://doi.org/10.1093/gbe/evz097
  9. Yu HY, Chu KH, Tsang LM, Ma KY. 2024. Incomplete lineage sorting and long-branch attraction confound phylogenomic inference of Pancrustacea. Frontiers in Ecology and Evolution 12:1243221. DOI 10.3389/fevo.2024.1243221. https://doi.org/10.3389/fevo.2024.1243221
  10. Du S, Tihelka E, Yu D, Chen W-J, Bu Y, Cai C, Engel MS, Luan Y-X, 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
  11. Höpel CG, Yeo D, Grams M, Meier R, Richter S. 2022. Mitogenomics supports the monophyly of Mysidacea and Peracarida (Malacostraca). Zoologica Scripta 51(5):603-613. DOI 10.1111/zsc.12554. https://doi.org/10.1111/zsc.12554
  12. Barta A-C, Grams M, Bracken-Grissom H, Brix S, Cordeiro LM, Cummings B, Collins S, Farris WJ, Gerken S, Höpel CG, Lörz A-N, McKim S, Meland K, Kruckenhauser L, Olesen J, Peres PA, Richter S, Wetzer R, Williams J, Kocot KM, Schwentner M. 2026. Phylogenomics supports monophyly of marsupial crustaceans: a journey to direct development. Molecular Biology and Evolution 43(8):msag178. DOI 10.1093/molbev/msag178. https://doi.org/10.1093/molbev/msag178
  13. Iwasa-Arai T, Linse K, Andrade SCS, Giribet G. 2025. Unpouching Peracarida relationships with ultraconserved elements. bioRxiv preprint. DOI 10.1101/2025.11.21.689751. https://doi.org/10.1101/2025.11.21.689751
  14. Gerken S, Błażewicz M, Kocot KM, Richter S, Schwentner M, Wetzer R. 2026. Phylogeny of the fascinating and frustrating Peracarida: the past, the present and the future. Arthropod Systematics & Phylogeny 84:549-563. DOI 10.3897/asp.84.e180526. https://doi.org/10.3897/asp.84.e180526
  15. Grams M, Torres A, Wirkner CS, Richter S. 2025. A new morphological phylogeny of Malacostraca comparing the application of character dependencies and implied weighting. Cladistics 41(3):283-303. DOI 10.1111/cla.12611. https://doi.org/10.1111/cla.12611
  16. Dreyer N, Bernot JP, Olesen J, Kolbasov GA, Høeg JT, Machida RJ, Chan BKK. 2025. Phylogenomics of enigmatic crustacean y-larvae reveals multiple origins of parasitism in barnacles. Current Biology 35(14):3356-3367.e4. DOI 10.1016/j.cub.2025.06.007. https://doi.org/10.1016/j.cub.2025.06.007