bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.182915; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 Lack of support for Deuterostomia prompts reinterpretation of the first Bilateria. 2 3 Authors and Affiliations 4 Paschalia Kapli1, Paschalis Natsidis1, Daniel J. Leite1, Maximilian Fursman1,&, Nadia Jeffrie1, 5 Imran A. Rahman2, Hervé Philippe3, Richard R. Copley4, Maximilian J. Telford1* 6 7 1. Centre for Life’s Origins and Evolution, Department of Genetics, Evolution and Environment, 8 University College London, Gower Street, London WC1E 6BT, UK 9 10 2. Oxford University Museum of Natural History, Parks Road, Oxford OX1 3PW, UK 11 3. Station d’Ecologie Théorique et Expérimentale, UMR CNRS 5321, Université Paul Sabatier, 12 09200 Moulis, France 13 4. Laboratoire de Biologie du Développement de Villefranche-sur-mer (LBDV), Sorbonne 14 Université, CNRS, 06230 Villefranche-sur-mer, France 15 & Current Address: Geology and Palaeoenvironmental Research Department, Institute of 16 Geosciences, Goethe University Frankfurt - Riedberg Campus, Altenhöferallee 1, 60438 17 Frankfurt am Main 18 19 20 21 22 23 *Correspondence: [email protected]. 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.182915; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 Abstract 25 The bilaterally symmetric animals (Bilateria) are considered to comprise two monophyletic 26 groups, Protostomia and Deuterostomia. Protostomia contains the Ecdysozoa and the 27 Lophotrochozoa; Deuterostomia contains the Chordata and the Xenambulacraria (Hemichordata, 28 Echinodermata and Xenacoelomorpha). Their names refer to a supposed distinct origin of the 29 mouth (stoma) in the two clades, but these groups have been differentiated by other 30 embryological characters including embryonic cleavage patterns and different ways of forming 31 their mesoderm and coeloms. Deuterostome monophyly is not consistently supported by recent 32 studies. Here we compare support for Protostomia and Deuterostomia using five recently 33 published, phylogenomic datasets. Protostomia is always strongly supported, especially by 34 longer and higher quality genes. Support for Deuterostomia is always equivocal and barely 35 higher than support for paraphyletic alternatives. Conditions that can cause tree reconstruction 36 errors - inadequate models, short internal branch, faster evolving genes, and unequal branch 37 lengths - correlate with statistical support for monophyletic deuterostomes. Simulation 38 experiments show that support for Deuterostomia could be explained by systematic error. A 39 survey of molecular characters supposedly diagnostic of deuterostomes shows many are not valid 40 synapomorphies. The branch between bilaterian and deuterostome common ancestors, if real, is 41 very short. This finding fits with growing evidence suggesting the common ancestor of all 42 Bilateria had many deuterostome characteristics. This finding has important implications for our 43 understanding of early animal evolution and for the interpretation of some enigmatic Cambrian 44 fossils such as vetulicolians and banffiids. 45 46 47 48 49 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.182915; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 50 Main 51 52 In the case of a burst of diversification such as is apparent at the base of the Cambrian, 53 reconstructing the evolutionary history of taxa is difficult. Limited time between speciations 54 (short internal branches) lead to the accumulation of a small amount of phylogenetic signal and 55 this signal is easily blurred by the numerous substitutions occurring later (long terminal 56 branches). These problems are exacerbated by heterogeneities in the evolutionary process which 57 can result in model violations that can cause systematic errors1. Very short internal branches also 58 strongly imply minimal evolutionary change meaning that common ancestors of nested clades 59 are expected to have been highly similar. 60 61 The deuterostome branch has weak support. 62 The different topologies relating Chordata, Xenambulacraria and Protostomia seen in recently 63 published animal phylogenies suggest that the support for monophyletic deuterostomes (Fig. 1a) 64 may be low, in contrast with the consistent, strong support for the monophyly of the protostomes. 65 To study this difference, we have gathered five recent, independently generated, phylogenomic 66 datasets covering the diversity of animal phyla (i.e., 2–6). We use these data to investigate the 67 support for different topologies relating the Chordata, Xenambulacraria, Ecdysozoa and 68 Lophotrochozoa. We first asked whether the branches leading to protostomes and deuterostomes 69 were of similar length, under the assumption that both are monophyletic. 70 71 Using a fixed topology taken from the original publications for each dataset (modified when 72 necessary to enforce monophyly of deuterostomes), we optimised branch lengths using the site- 73 homogeneous LG+F+G model. In Figure 1b we see that, while the overall amount of change in 74 branches leading from the bilaterian common ancestor to both protostomes and deuterostomes 75 differs between datasets, in all cases, the branch leading to the protostomes is approximately 76 twice the length of the branch leading to the deuterostomes. This is a conservative measure of the 77 difference, as better fitting models (see later) give longer estimates for the protostome branch 78 and shorter estimates for the deuterostome branch (for the Laumer dataset the protostome branch 79 is estimated as 1.75 times longer than deuterostome under LG and 5.7 times longer under CAT- 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.182915; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 80 LG). This difference between protostome and deuterostome branches is reinforced when we look 81 at the change in log likelihood (∆lnL) between a fully resolved tree compared to trees in which 82 either the protostome or deuterostome branch is collapsed into a polytomy. The ∆lnL observed 83 on collapsing the branch leading to protostomes is considerably greater for all five datasets than 84 the ∆lnL when the deuterostome branch is collapsed (Fig. 1c), showing considerably less signal 85 supporting deuterostomes than protostomes. 86 87 To get a first indication of whether this weaker support for deuterostomes results from conflict 88 between genes or from a lack of signal across genes and across datasets, we repeated these 89 measures of branch lengths and ∆lnL on individual genes in all five datasets (Fig. 1d,e). We find 90 that, for the great majority of genes across all datasets, as for the concatenated datasets, there is a 91 longer branch leading to the protostomes than to deuterostomes (P longer: 3376 (70%); D longer: 92 1402 (29%), Supplementary Data 1) and consistently stronger support (larger ∆lnL) for 93 protostomes than for deuterostomes (∆lnL P larger: 3312 (68.6%); ∆lnL D larger: 1345 (27.8%), 94 Supplementary Data 2). Most genes support monophyletic deuterostomes much less strongly 95 than they support protostomes. 96 97 Minority of genes support deuterostome monophyly. 98 While the difference in branch lengths and likelihood indicate weaker evidence for the 99 monophyly of deuterostomes across different genes relative to the support for protostomes, the 100 measures presented are based on trees in which deuterostomes were constrained to be 101 monophyletic, meaning that we could not show any topological conflict between genes. To 102 measure conflict between genes we considered each gene individually and compared the 103 lnLikelihood of a tree supporting protostome monophyly (“PM”) or deuterostome monophyly 104 (“DM”) with the lnLikelihoods of two alternative topologies for each of these two clades: 105 Protostome Paraphyly “P1”: Lophotrochozoa sister to Deuterostomia; Protostome Paraphyly 106 “P2”: Ecdysozoa sister to Deuterostomia; Deuterostome Paraphyly “D1”: Xenambulacraria sister 107 to Protostomia; Deuterostome Paraphyly “D2”: Chordata sister to Protostomia. (see Fig. 2). 108 We visualised the relative lnLikelihoods for the three topologies for every gene on a triangular 109 plot (Fig. 2). For the protostome trees, most genes have a strong preference for a single topology 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.07.01.182915; this version posted July 2, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 110 (77.31% on average