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Letter doi:10.1038/nature16520 Xenacoelomorpha is the sister group to Nephrozoa Johanna Taylor Cannon1, Bruno Cossermelli Vellutini2, Julian Smith III3, Fredrik Ronquist1, Ulf Jondelius1 & Andreas Hejnol2 The position of Xenacoelomorpha in the tree of life remains a major unresolved question in the study of deep animal relationships1. Xenacoelomorpha, comprising Acoela, Nemertodermatida, and Xenoturbella, are bilaterally symmetrical marine worms that lack several features common to most other bilaterians, for example an anus, nephridia, and a circulatory system. Two conflicting hypotheses are under debate: Xenacoelomorpha is the sister group to all remaining Bilateria (= Nephrozoa, namely protostomes and deuterostomes)2,3 or is a clade inside Deuterostomia4. Thus, determining the phylogenetic position of this clade is pivotal for understanding the early evolution of bilaterian features, or as a case of drastic secondary loss of complexity. Here we show robust phylogenomic support for Xenacoelomorpha as the sister taxon of Nephrozoa. Our phylogenetic analyses, based on 11 novel xenacoelomorph transcriptomes and using different models of evolution under maximum likelihood and Bayesian inference analyses, strongly corroborate this result. Rigorous testing of 25 experimental data sets designed to exclude data partitions and taxa potentially prone to reconstruction biases indicates that longbranch attraction, saturation, and missing data do not influence these results. The sister group relationship between Nephrozoa and Xenacoelomorpha supported by our phylogenomic analyses implies that the last common ancestor of bilaterians was probably a benthic, ciliated acoelomate worm with a single opening into an epithelial gut, and that excretory organs, coelomic cavities, and nerve cords evolved after xenacoelomorphs separated from the stem lineage of Nephrozoa. Acoela have an essential role in hypotheses of bilaterian body plan evolution5. Acoels have been compared to cnidarian planula larvae because they possess characters such as a blind gut, a net-like nervous system, and they lack nephridia. However, they also share apomorphies with Bilateria such as bilateral symmetry and a mesodermal germ layer that gives rise to circular and longitudinal muscles. Classic systematics placed acoels in Platyhelminthes6, or as a separate early bilaterian lineage7,8. When nucleotide sequence data became available, Acoela were placed as the sister group of Nephrozoa9. Nemertodermatida were originally classified within Acoela, but were soon recognized as a separate clade on morphological grounds10. Subsequently, nucleotide sequence data fuelled a debate on whether nemertodermatids and acoels form a monophyletic group, the Acoelomorpha, or if nemertodermatids and acoels are independent early bilaterian lineages as suggested by several studies, for example refs 11 and 12. The enigmatic Xenoturbella was first placed together with Acoela and Nemertodermatida13,14; then an ultrastructural appraisal supported its position as sister group of all other bilaterians15. The first molecular study suggested Xenoturbella to be closely related to molluscs16, whereas other analyses proposed a deuterostome affiliation17,18. Recent analyses of molecular data reunited Xenoturbella with acoels and nemertodermatids2–4 to form a clade called Xenacoelomorpha (Fig. 1a). Current conflicting hypotheses suggest that Xenacoelomorpha are the sister group of Deuterostomia4, are nested within Deuterostomia4, are the sister group of Nephrozoa2,3, or are polyphyletic, with Xenoturbella included within Deuterostomia and the Acoelomorpha as sister taxon to remaining Bilateria19 (Fig. 1b–e). The deuterostome affiliation derives support from three lines of evidence4: an analysis of mitochondrial gene sequences, microRNA complements, and a phylogenomic data set. Analyses of mitochondrial genes recovered Xenoturbella within deuterostomes18. However, limited mitochondrial data (typically ~16 kilobase total nucleotides, 13 protein-coding genes) are less efficient in recovering higher-level animal relationships than phylogenomic approaches, especially in long-branching taxa1. The one complete and few partial mitochondrial genomes for acoelomorphs are highly divergent in terms of both gene order and nucleotide sequence19,20. Analyses of new complete mitochondrial genomes of Xenoturbella spp. do not support any phylogenetic hypothesis for this taxon21. Ref. 4 proposes that microRNA data support Xenacoelomorpha within the deuterostomes; however, microRNA distribution is better explained by a sister relationship between Xenacoelomorpha and Nephrozoa both under parsimony4,22 and under Bayesian inference22. Phylogenomic analyses recovering xenacoelomorph taxa within Deuterostomia show branching patterns that differ significantly a Xenacoelomorpha Nemertodermatida b Bilateria Nephrozoa Deuterostomia d Bilateria Deuterostomia Xenoturbella Acoela c Xenacoelomorpha Protostomia Protostomia Chordata Chordata Bilateria Deuterostomia Ambulacraria Xenambulacraria Protostomia Xenacoelomorpha Chordata Ambulacraria e Xenacoelomorpha Ambulacraria Acoelomorpha Bilateria Nephrozoa Deuterostomia Xenambulacraria Protostomia Chordata Xenoturbella Ambulacraria Figure 1 | Phylogenetic hypotheses concerning Xenacoelomorpha from previous molecular studies. a, Relationships among Xenacoelomorpha. Xenoturbella is sister to Acoelomorpha (Acoela + Nemertodermatida). Illustrated species from left to right: Flagellophora apelti, Diopisthoporus psammophilus, X. bocki. b, Xenacoelomorpha is sister taxon to Nephrozoa (phylogenomic analyses2,3). c, Xenacoelomorpha is sister taxon to Ambulacraria within deuterostomes (phylogenomic analyses4). d, Xenacoelomorpha is sister taxon to Ambulacraria + Chordata (mitochondrial protein analyses4,19). e, Xenoturbella is within Deuterostomia, while Acoelomorpha form two separate clades outside Nephrozoa (molecular systematic analyses11), or its sister group (some mitochondrial protein analyses19). Colours in b–e indicate Xenacoelomorpha (red), Protostomia (blue), Deuterostomia (green). 1 Naturhistoriska Riksmuseet, PO Box 50007, SE-104 05 Stockholm, Sweden. 2Sars International Centre for Marine Molecular Biology, University of Bergen, Thormøhlensgate 55, 5008 Bergen, Norway. 3Department of Biology, Winthrop University, 701 Oakland Avenue, Rock Hill, South Carolina 29733, USA. 4 f e b r u a r y 2 0 1 6 | V O L 5 3 0 | N A T URE | 8 9 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter Xenoturbella bocki Bilateria 47/65/47 78/85/78 71/60/71 99/ 100/ 99 98/98/98 66/78/66 39/43/39 70/ 74/ 70 95/99/95 87/83/87 55/53/55 BS support Schmidtea mediterranea Schistosoma mansoni Taenia pisiformes Prostheceraeus vittatus Macrostomum lignano Lepidodermella squamata Megadasys sp. Macrodasys sp. Membranipora membranacea Bryozoa Loxosoma pectinaricola Entoprocta Barentsia gracilis Gastrotricha Deuterostomia Botryllus schlosseri Ciona intestinalis Chordata Homo sapiens Gallus gallus Petromyzon marinus Dumetocrinus sp. 67/--/67 Labidiaster annulatus 77/52/77 Astrotomma agassizi Ambulacraria Leptosynapta clarki 86/76/86 (Echinodermata Strongylocentrotus purpuratus Cephalodiscus gracilis +Hemichordata) 67/--/67 Saccoglossus mereschkowskii Ptychodera bahamensis Schizocardium braziliense Eunicella cavolinii Nematostella vectensis Acropora digitifera Cnidaria Stomolophus meleagris Craspedacusta sowerby Agalma elegans Trichoplax adhaerens Placozoa Oscarella carmela Leucosolenia complicata Sycon ciliatum Aphrocallistes vastus Porifera Cliona varians Amphimedon queenslandica Mnemiopsis leidyi Pleurobrachia bachei Ctenophora Euplokamis dunlapae Monosiga brevicollis Choanoflagellata Salpingoeca rosetta 95/99/95 89/97/89 Branchiostoma floridae Spiralia Platyhelminthes 96/100/96 Nephrozoa Priapulus caudatus Priapulida Ecdysozoa Halicryptus spinulosus Peripatopsis capensis Onychophora Drosophila melanogaster Daphnia pulex Arthropoda Strigamia maritima Ixodes scapularis Helobdella robusta Pomatoceros lamarckii Annelida Capitella teleta Phoronis psammophila Terebratalia transversa Lophophorata Hemithiris psittacea (Phoronida + Brachiopoda) Novocrania anomala Lineus longissimus Cephalothrix hongkongiensis Nemertea Leptochiton rugatus Crassostrea gigas Mollusca Lottia gigantea Adineta ricciae Adineta vaga Rotifera Brachionus calyciflorus Acoela 63/76/63 Xenacoelomorpha Diopisthoporus gymnopharyngeus Diopisthoporus longitubus Hofstenia miamia Isodiametra pulchra Eumecynostomum macrobursalium Convolutriloba macropyga 92/96/92 Childia submaculatum Ascoparia sp. Meara stichopi Nemertodermatida Sterreria sp. Nemertoderma westbladi =100% all models, ProtTest/LG4X/LG 0.2 Figure 2 | Maximum likelihood topology of metazoan relationships inferred from 212 genes. Maximum likelihood tree is shown as inferred using the best-fitting amino-acid substitution model for each gene. Bootstrap support values from analyses inferred under alternative models of amino-acid substitution are indicated at the nodes (best-fitting model for each orthologous group selected by ProtTest/LG4X across all partitions/LG + I + Γ across all partitions, 100 bootstrap replicates). Filled blue circles represent 100% bootstrap support under all models of evolution. Species indicated in bold are new transcriptomes published with this study. between alternative models of evolution4. Conflicting results in studies that used the same expressed sequence tag data for xenacoelomorphs2,4 suggest some degree of model misspecification, missing data generating positively misleading signal, or long-branch attraction (LBA) in either or both of these studies. Testing of hypotheses under alternative models of evolution, data set partitioning, and taxon selection schemes can identify possible weaknesses of a data set. Here, we use this approach to test the phylogenetic position of Acoela, Nemertodermatida, and Xenoturbella. Novel Illumina RNaseq data were collected for six acoel species, four nemertodermatids, Xenoturbella bocki, and six additional diverse metazoans (Supplementary Table 1). Acoel and nemertoder­ matid species were selected to broadly represent the diversity of these two clades, including two representatives of the earliest-branching clade of Acoela, Diopisthoporidae 23 . With the exception of Hofstenia miamia in ref. 3, previous phylogenomic analyses of acoels have included only representatives of Convolutidae and Isodiametridae, which possess several highly derived morphological characters. Our data sets include 76 diverse metazoan taxa and 2 choanoflagellate outgroups (Supplementary Table 1). Our primary data set consists of 212 orthologous groups, 44,896 amino-acid positions, and 31% missing data (Extended Data Table 1). 9 0 | N A T URE | V O L 5 3 0 | 4 f e b r u a r y 2 0 1 6 © 2016 Macmillan Publishers Limited. All rights reserved Letter RESEARCH Xenoturbella bocki 0.99 0.99 0.83 0.78 Diopisthoporus longitubus Diopisthoporus gymnopharyngeus Hofstenia miamia Isodiametra pulchra Eumecynostomum macrobursalium Childia submaculatum Convolutriloba macropyga Nemertoderma westbladi Sterreria sp. Meara stichopi Xenacoelomorpha Ascoparia sp. Priapulus caudatus Ecdysozoa Halicryptus spinulosus Peripatopsis capensis Ixodes scapularis Strigamia maritima Daphnia pulex Drosophila melanogaster Leptochiton rugatus Lottia gigantea Crassostrea gigas Phoronis psammophila Novocrania anomala Terebratalia transversa Hemithiris psittacea Helobdella robusta Pomatoceros lamarckii Capitella teleta Lineus longissimus Cephalothrix hongkongiensis Megadasys sp. Macrodasys sp. Lepidodermella squamata Membranipora membranacea Schmidtea mediterranea Taenia pisiformes Schistosoma mansoni 0.51 0.88 Spiralia Prostheceraeus vittatus Macrostomum lignano Loxosoma pectinaricola Barentsia gracilis Brachionus calyciflorus Adineta vaga Adineta ricciae Petromyzon marinus Deuterostomia Homo sapiens Gallus gallus Ciona intestinalis Botryllus schlosseri Branchiostoma floridae Saccoglossus mereschkowskii Schizocardium c.f. braziliense Ptychodera bahamensis Cephalodiscus gracilis Dumetocrinus sp. Strongylocentrotus purpuratus Leptosynapta clarki Astrotomma agassizi 0.94 Labidiaster annulatus Eunicella cavolinii Nematostella vectensis Acropora digitifera Cnidaria Stomolophus meleagris Craspedacusta sowerby Agalma elegans Trichoplax adhaerens Pleurobrachia bachei Mnemiopsis leidyi Ctenophora Euplokamis dunlapae Oscarella carmela Sycon ciliatum Leucosolenia complicata Porifera Aphrocallistes vastus Cliona varians Amphimedon queenslandica Monosiga brevicollis Salpingoeca rosetta 0.3 Figure 3 | Bayesian inference topology of metazoan relationships inferred from 212 genes under the CAT + GTR + Γ model. Filled blue circles indicate posterior probabilities of 1.0. Shown is the majority rule consensus tree of two independent chains of > 17,000 cycles each and burn-in of 5,000 cycles. Convergence of the two chains was indicated by a ‘maxdiff ’ value of 0.25. Position of Xenacoelomorpha was unchanged in two additional independent chains, which did not converge with the chains shown above owing to alternative positions of Trichoplax adhaerens and Membranipora membranacea. Sequences were taken entirely from Illumina transcriptomes or predicted transcripts from genomic data. Gene occupancy per taxon ranged from 100% for Homo sapiens and Drosophila melanogaster to 8% for the nemertodermatid Sterreria sp., with median per-taxon gene occupancy of 90% and an average of 80% (Supplementary Table 2). Notably, gene coverage for key taxa is enhanced over previous phylogenomic analyses: X. bocki, six acoels, and two nemertodermatids have > 90% gene occupancy in our 212 orthologous group data set, whereas the best represented acoelomorph terminal in ref. 4 had an occupancy of 63%. Maximum likelihood analyses were conducted under the bestfitting model for each individual gene partition, or the LG model, or the LG4X model24 over each independent partition. The LG4X model is composed of four substitution matrixes designed to improve modelling of site heterogeneity24. Bayesian analyses were conducted with the site-heterogeneous CAT + GTR + Γ model and GTR + Γ. To further validate the robustness of our results to variations in substitution model specification, we performed Bayesian inference analyses under an independent substitution model using a back-translated nucleotide data set derived from our amino-acid alignment. To test whether any 4 f e b r u a r y 2 0 1 6 | V O L 5 3 0 | N A T URE | 9 1 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter a Acoela Xenacoelomorpha Nemertodermatida Xenoturbella Arthropoda Ecdysozoa Onychophora Priapulida Mollusca Bilateria Brachiopoda Trochozoa Protostomia Phoronida Annelida Nemertea Platyhelminthes Entoprocta Planulozoa Nephrozoa Spiralia Gastrotricha Bryozoa Rotifera Parahoxozoa Deuterostomia Ambulacraria Chordata Cnidaria Metazoa Placozoa Ctenophora Porifera Choanoflagellata = Maximal support in analyses of 212, 336, and 881 genes >99% b >70% S ep acith lik el e ia lg N ut er ve -n et M es od er m Bi la te ra lit y N ep hr id ia particular taxon was biasing our analyses owing to artefacts such as LBA, we conducted a series of taxon-pruning experiments. Additional data sets were analysed that minimized missing data, excluded taxa and individual genes identified to be potentially more subject to LBA artefacts, and genes or positions that were more saturated. Using our standard pipeline, for the best-sampled 56 taxa, we also generated a data set with 336 orthologous groups, 81,451 amino acids, and 11% missing data. Lastly, using an independent pipeline for orthologous gene selection, we generated a set of 881 orthologous groups. This larger data set contained 77 operational taxonomic units, 337,954 aminoacid positions, and 63% matrix occupancy. In all, we generated 25 unique data matrixes to address the robustness of phylogenetic signal and sensitivity of our results to parameter changes (Extended Data Table 1). Our analyses consistently supported monophyletic Xenacoelo­ morpha as sister group of Nephrozoa (Figs 2–4, Extended Data Figs 1–4 and Extended Data Table 1). Within Xenacoelomorpha, Xenoturbella is the sister taxon of Acoela + Nemertodermatida. Maximum likelihood analyses under all models (Fig. 2), Bayesian analyses under the site-heterogeneous CAT + GTR + Γ model (Fig. 3), as well as analyses of back-translated nucleotides (Extended Data Fig. 5) all recover this topology. We found no evidence of LBA influencing the position of Xenacoelomorpha or any other group in the tree. Differing outgroup schemes do not affect the position of Xenacoelomorpha (Supplementary Figs 4–9); neither does exclusion of taxa or genes more subject to LBA (Supplementary Figs 14–17). Monophyletic Deuterostomia (excluding Xenoturbella), Ecdysozoa, and Spiralia are robustly recovered, with Ctenophora as the earliest branching metazoan in all maximum likelihood analyses, while Porifera holds this position in Bayesian analyses under the CAT + GTR + Γ model (Fig. 3). Taxon-exclusion analyses, where Acoelomorpha alone (Supplementary Fig. 1) or Xenoturbella alone (Extended Data Fig. 3) were included, recovered these taxa as the first branch of Bilateria. Approximately unbiased tests strongly reject the alternative hypothesis constraining Xenacoelomorpha within Deuterostomia. Leaf stability indices for all taxa in the primary 212 orthologous group analysis were > 97% (Supplementary Table 2), suggesting that improved matrix and taxon coverage in our analyses had a positive effect on overall taxon stability compared with ref. 25, where both included acoels had leaf stability indices of 78%. In our own calculations of leaf stability index from the data set of ref. 4, the six representative xenacoelomorph species have the six lowest leaf stabilities of all included taxa, ranging from 88% to 79% (Supplementary Table 3). To assess gene conflict, we conducted decomposition analyses using ASTRAL26, which calculates the species tree that agrees with the largest number of quartets derived from each gene tree and their respective bootstrap replicates (Extended Data Fig. 4). This analysis finds strong support for the position of Xenacoelomorpha (bootstrap 99%). Refs 27 and 28 pointed to issues with incongruence in phylogenomic analyses of ribosomal protein genes versus other protein-coding genes. Notably, in our 212 orthologous group set, only five ribosomal protein genes were retained after screening for paralogous groups. To investigate if this gene class may have biased previous results, we generated an additional data matrix composed of 52 ribosomal protein genes that passed through our other filters for gene length and taxon presence. In maximum likelihood analyses of this data set, Xenacoelomorpha, Acoelomorpha, Nemertodermatida, Deuterostomia and Spiralia are all non-monophyletic (Supplementary Fig. 21). Ribosomal protein genes are heavily represented in the xenacoelomorph data in previous studies, comprising > 50% of the gene occupancy in most cases. Gene partition information was not made available for the study proposing a deuterostome position for Xenacoelomorpha4, so re-analysis of the data without ribosomal protein genes was not possible. We suggest that insufficient data for key taxa and a reliance on ribosomal protein genes were biasing the results, causing Xenacoelomorpha to group within Deuterostomia. Cnidaria Xenacoelomorpha Bilateria Nephrozoa Protostomia Deuterostomia Figure 4 | Summary of metazoan relationships as inferred in this study. a, Summary of phylogenomic results based on analyses of 212, 336, and 881 genes. Xenacoelomorpha is a monophyletic clade sister to Nephrozoa with > 99% support in all analyses. b, Interrelationships among four major animal clades, Cnidaria, Xenacoelomorpha, Protostomia, and Deuterostomia, with selected morphological characters mapped onto the tree as ancestral states for each of the four clades. Within Xenacoelomorpha, morphological complexity differs among the three groups, as should be expected in a clade of the same age as Nephrozoa. The simplest organization is evident in Xenoturbella, with a sac-like epithelial gut opening to a simple mouth, a basiepidermal nervous system, and no gonopores or secondary reproductive organs13. Nemertodermatida also have an epithelial gut, but the mouth appears to be a transient structure10. Furthermore, the position and anatomy of the nervous system and the male copulatory organ are variable. The more than 400 nominal species of Acoela (compared with 18 nemertodermatids and 5 Xenoturbella species) exhibit considerable morphological variation: acoels have no intestinal lumen although a mouth opening and sometimes a pharynx is present23. The nervous system is highly variable, there are one or two gonopores, and often accessory reproductive organs23. The morphological evolution that occurred within Xenacoelomorpha provides an interesting parallel case to Nephrozoa. The sister group relationship between Xenacoelomorpha and Nephrozoa allows us to infer the order in which bilaterian features were evolved12,29. The bilaterian ancestor was probably a soft-bodied, small ciliated benthic worm5,23,29,30. Mesoderm and body axis were established before the split between Xenacoelomorpha and Nephrozoa, whereas nephridia evolved in the stem lineage of nephrozoans (Fig. 4). Centralization of the nervous system appears to have evolved in parallel in the Xenacoelomorpha and Nephrozoa. Further investigations of the genomic architecture and biology of xenacoelomorphs will provide insights into molecular, developmental, and cellular building blocks used for evolving complex animal body plans and organ systems. 9 2 | N A T URE | V O L 5 3 0 | 4 f e b r u a r y 2 0 1 6 © 2016 Macmillan Publishers Limited. 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Error, signal, and the placement of Ctenophora sister to all other animals. Proc. Natl Acad. Sci. USA 112, 5773–5778 (2015). 29. Hejnol, A. & Martindale, M. Q. Acoel development supports a simple planula-like urbilaterian. Phil. Trans. R. Soc. B 363, 1493–1501 (2008). 30. Laumer, C. E. et al. Spiralian phylogeny informs the evolution of microscopic lineages. Curr. Biol. 25, 2000–2006 (2015). Supplementary Information is available in the online version of the paper. Acknowledgements The Swedish Research Council provided funding for U.J. and J.T.C. (grant 2012-3913) and F.R. (grant 2014-5901). A.H. received support from the Sars Core budget and Marie Curie Innovative Training Networks ‘NEPTUNE’ (FP7-PEOPLE-2012-ITN 317172) and FP7-PEOPLE-2009-RG 256450. We thank N. Lartillot and K. Kocot for discussions. Hejnol laboratory members K. Pang and A. Børve assisted with RNA extraction; A. Boddington, J. Bengtsen and A. Elde assisted with culture for Isodiametra pulchra and Convolutriloba macropyga. Thanks to W. Sterrer for collection of Sterreria sp. and Ascoparia sp., and to R. Janssen for finding X. bocki. The Sven Lovén Centre of Marine Sciences Kristineberg, University of Gothenburg, and the Interuniversity Institute of Marine Sciences in Eilat provided logistical support for field collection. S. Baldauf assisted with laboratory space and resources for complementary DNA synthesis. We thank K. Larsson for the original illustrations. Computations were performed on resources provided by the Swedish National Infrastructure for Computing (SNIC). Transcriptome assembly, data set construction, RAxML and PhyloBayes analyses were performed using resources provided through Uppsala Multidisciplinary Center for Advanced Computational Science (UPPMAX) under project b2013077, and MrBayes analyses were run under project snic2014-1-323. Author Contributions J.T.C., U.J., B.C.V., and A.H. conceived and designed the study. U.J. and A.H. collected several specimens and J.S. III collected Diopisthoporus gymnopharyngeus specimens. J.T.C. and B.C.V. performed molecular work and RNA sequencing assembly. J.T.C. assembled the datasets and performed phylogenetic analyses. F.R. conducted Bayesian phylogenetic analyses using MrBayes. All authors contributed to writing the manuscript. Author Information Sequence data have been deposited in the NCBI Sequence Read Archive under BioProject PRJNA295688. Data matrices and trees from this study are available from the Dryad Digital Repository (http://datadryad.org) under DOI 10.5061/dryad.493b7. Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests. Readers are welcome to comment on the online version of the paper. Correspondence and requests for materials should be addressed to J.T.C. (joie.cannon@gmail.com) or A.H. (andreas.hejnol@uib.no). 4 f e b r u a r y 2 0 1 6 | V O L 5 3 0 | N A T URE | 9 3 © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter Methods No statistical methods were used to predetermine sample size. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Molecular methods and sequencing. We generated novel RNA-seq data from six acoels, four nemertodermatids, X. bocki, and six additional diverse metazoans (Supplementary Table 1). Total RNA was extracted from fresh or RNAlater (Ambion) preserved specimens using TRI Reagent Solution (Ambion) or the RNeasy Micro Kit (Qiagen), prepared using the SMART complementary DNA library construction kit (Clontech), and sequenced as 2 × 100 paired end runs with Illumina HiSeq 2000 at SciLifeLab (Stockholm, Sweden) or GeneCore (EMBL Genomics Core Facilities). Illumina data were supplemented with publically available RNaseq and genome data (Supplementary Table 1) to generate a final data set including 76 diverse metazoans and 2 choanoflagellate outgroup taxa. Data set assembly. Both novel RNA-seq data and raw Illumina sequences taken from the NCBI Sequence Read Archive were assembled using Trinity31. Assembled data were translated using Transdecoder (http://transdecoder.sf.net). To determine orthologous genes, we used two methods: a more restrictive and standard approach using HaMStR (Hidden Markov Model based Search for Orthologues using Reciprocity)32, as well as an approach designed to generate a broader set of genes for phylogenetic inference, using the software ProteinOrtho33. Protocols for gene selection using HaMStR followed refs 34 and 35. Translated unigenes for all taxa were searched against the model organisms core orthologue set of HaMStR using the strict option and D. melanogaster as the reference taxon. Sequences shorter than 50 amino acids were deleted, and orthologous groups sampled for fewer than 30 taxa were excluded to reduce missing data. To trim mistranslated ends, if one of the first or last 20 characters of sequences was an X, all characters between that X and the end of the sequence were removed. The orthologous groups were then aligned using MAFFT36 and trimmed using Aliscore37 and Alicut (https://www.zfmk.de/en/research/research-centres-and-groups/utilities). At this stage, sequences that were greater than 50% gaps and alignments shorter than 100 amino acids were discarded. To remove potentially paralogous genes, we generated single gene trees using FastTree38 and filtered these using PhyloTreePruner39. For 78 taxa, this protocol retained 212 orthologous groups, 44,896 amino acids, with 31% missing data. This protocol was repeated with the 56 taxa with highest percentage of gene coverage, resulting in a data matrix of 336 genes, 81,451 amino acids, and 11% missing data. To generate the ProteinOrtho data set, Sterreria sp. was excluded owing to its small library size. Translated assemblies were filtered to remove mistranslated ends as described above, and only sequences longer than 50 amino acids were retained for clustering in ProteinOrtho. In ProteinOrtho, we used the steps option, the default E-value for BLAST, and minimum coverage of best BLAST alignments of 33%. Resulting clusters were filtered to include only putative orthologous groups containing greater than 40 taxa, then aligned as above with MAFFT. For each alignment a consensus sequence was inferred using the EMBOSS program infoalign40. Infoalign’s ‘change’ calculation computes the percentage of positions within each sequence in each alignment that differ from the consensus. Sequences with a ‘change’ value larger than 75 were deleted, helping to exclude incorrectly aligned sequences. orthologous groups were then realigned with MAFFT, trimmed with Aliscore and Alicut, and processed as above. After filtering for paralogous groups with PhyloTreePruner, 881 orthologous groups were retained. Owing to the smaller size of the data set and amount of computational resources required, taxon pruning and signal dissection analyses were performed solely on the primary HaMStR gene set. For taxon exclusion experiments, individual orthologous group alignments were realigned using MAFFT following the removal of selected taxa. TreSpEx41 was used to assess potential sources of misleading signal, including standard deviation of branch-length heterogeneity (LB) and saturation. Sites showing evidence of saturation and compositional heterogeneity were removed using Block Mapping and Gathering with Entropy (BMGE)42, using the ‘fast’ test of compositional heterogeneity (-s FAST) and retaining gaps (-g 1). Phylogenetic analysis. Maximum likelihood analyses of the complete 212 orthologous group data matrix were performed using RAxML version 8.0.20-mpi43 under the best-fitting models for each gene partition determined by ProtTest version 3.4 (ref. 44). The best fitting model for all but 3 of the 212 orthologous groups was LG, so further maximum likelihood analyses were performed using the PROTGAMMAILG option. Bootstrapped trees from the 212-gene data set were used to calculate leaf stability indices of each operational taxonomic unit using the Roguenarok server (http://www.exelixis-lab.org/). Bayesian analyses were conducted using PhyloBayes-MPI45 version 1.5a under the CAT + GTR + Γ model or GTR + Γ with four independent chains per analysis. Analyses ran for >12,000 cycles, until convergence of at least two chains was reached as assessed by maxdiff. Further Bayesian analyses were conducted in MrBayes version 3.2 (ref. 46). For the MrBayes analyses, we back-translated the aligned amino-acid data to nucleotides for first and second codon positions using the universal genetic code. Third codon position data were ignored. When the back translation was ambiguous, we preserved the ambiguity in the nucleotide data. For instance, serine is coded by TC{A, C, G, T} or AG{T, C}, where {…} denotes alternative nucleotides for a single codon site. Thus, for Serine the back translation is {A,T}{C, G}. This is the only back translation that is ambiguous both for the first and for the second codon positions. The back translation for arginine and leucine are also ambiguous but only for the first codon position. All other back translations are unambiguous for both the first and second codon sites. Thus, the back translation of first and second codon sites results in negligible information loss compared with the original nucleotide data. We analysed the resulting nucleotide data in MrBayes 3.2.6-svn(r1037)46 using a model with two partitions: one for first codon positions and one for second codon positions. For each partition we employed an independent substitution model, modelling rate variation across sites using a discrete gamma distribution (four categories) with a proportion of invariable sites (‘lset rates = invgamma’), and nucleotide substitutions with independent stationary state frequencies and a reversiblejump approach to the partitioning of exchangeability rates (‘lset nst = mixed’). We also uncoupled the partition rates (‘prset ratepr = variable’). All other settings were left at their defaults. For each analysis, we used four independent runs with four Metropolis-coupled chains each and ran them for 4,000,000 generations, sampling every 500 generations (‘mcmcp nrun = 4 nch = 4 ngen = 4000000 samplefreq = 500’). The analyses finished with an average standard deviation of split frequencies of 0.033 or less, and a potential scale reduction factor of 1.003 or less. The MrBayes data files and run scripts are provided at the Dryad Digital Repository. We additionally used ASTRAL26 to calculate an optimal bootstrapped species tree from individual RAxML gene trees decomposed into quartets. 31. Grabherr, M. G.et al. Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nature Biotechnol. 29, 644–652 (2011). 32. Ebersberger, I., Strauss, S. & von Haeseler, A. HaMStR: profile hidden markov model based search for orthologs in ESTs. BMC Evol. Biol. 9, 157 (2009). 33. Lechner, M. et al. Proteinortho: detection of (co-)orthologs in large-scale analysis. BMC Bioinformatics 12, 124 (2011). 34. Kocot, K. M. et al. Phylogenomics reveals deep molluscan relationships. Nature 477, 452–456 (2011). 35. Cannon, J. T. et al. Phylogenomic resolution of the hemichordate and echinoderm clade. Curr. Biol. 24, 2827–2832 (2014). 36. Katoh, K., Kuma, K., Toh, H. & Miyata, T. MAFFT version 5: improvement in accuracy of multiple sequence alignment. Nucleic Acids Res. 33, 511–518 (2005). 37. Misof, B. & Misof, K. A Monte Carlo approach successfully identifies randomness in multiple sequence alignments: a more objective means of data exclusion. Syst. Biol. 58, 21–34 (2009). 38. Price, M. N., Dehal, P. S. & Arkin, A. P. FastTree 2—approximately maximumlikelihood trees for large alignments. PLoS One 5, e9490 (2010). 39. Kocot, K. M., Citarella, M. R., Moroz, L. L. & Halanych, K. M. PhyloTreePruner: a phylogenetic tree-based approach for selection of orthologous sequences for phylogenomics. Evol. Bioinform. Online 9, 429–435 (2013). 40. Rice, P., Longden, I. & Bleasby, A. EMBOSS: the European Molecular Biology Open Software Suite. Trends Genet. 16, 276–277 (2000). 41. Struck, T. H. TreSpEx-Detection of misleading signal in phylogenetic reconstructions based on tree information. Evol. Bioinform. Online 10, 51–67 (2014). 42. Criscuolo, A. & Gribaldo, S. BMGE (Block Mapping and Gathering with Entropy): a new software for selection of phylogenetic informative regions from multiple sequence alignments. BMC Evol. Biol. 10, 210 (2010). 43. Stamatakis, A. RAxML version 8: a tool for phylogenetic analysis and post-analysis of large phylogenies. Bioinformatics 30, 1312–1313 (2014). 44. Darriba, D., Taboada, G. L., Doallo, R. & Posada, D. ProtTest 3: fast selection of best-fit models of protein evolution. Bioinformatics 27, 1164–1165 (2011). 45. Lartillot, N., Rodrigue, N., Stubbs, D. & Richer, J. PhyloBayes MPI: phylogenetic reconstruction with infinite mixtures of profiles in a parallel environment. Syst. Biol. 62, 611–615 (2013). 46. Ronquist, F. et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst. Biol. 61, 539–542 (2012). © 2016 Macmillan Publishers Limited. All rights reserved Letter RESEARCH Xenoturbella bocki XENACOELOMORPHA Meara stichopi Nemertoderma westbladi Isodiametra pulchra Childia submaculatum Convolutriloba macropyga Diopisthoporus longitubus Hofstenia miamia Drosophila melanogaster Daphnia pulex Strigamia maritima Ixodes scapularis ECDYSOZOA Halicryptus spinulosus Priapulus caudatus Capitella teleta SPIRALIA Helobdella robusta Lineus longissimus 72 Phoronis psammophila 80 Novocrania anomala Hemithiris psittacea 96 Terebratalia transversa Leptochiton rugatus Crassostrea gigas Lottia gigantea Brachionus calyciflorus Adineta vaga Adineta ricciae 56 98 Macrodasys sp. Macrostomum lignano 56 Prostheceraeus vittatus 98 Schistosoma mansoni Lepidodermella squamata Membranipora membranacea Dumetocrinus sp. DEUTEROSTOMIA Strongylocentrotus purpuratus Saccoglossus mereschkowskii Ptychodera bahamensis Homo sapiens Gallus gallus 44 Botryllus schlosseri 90 Ciona intestinalis Branchiostoma floridae Trichoplax adhaerens Stomolophus meleagris 64 Agalma elegans Craspedacusta sowerby CNIDARIA Nematostella vectensis Aphrocallistes vastus Cliona varians 44 Amphimedon queenslandica Oscarella carmela PORIFERA Sycon ciliatum Leucosolenia complicata Mnemiopsis leidyi Salpingoeca rosetta CTENOPHORA Monosiga brevicollis 0.2 Extended Data Figure 1 | Maximum likelihood topology of metazoan relationships inferred from 336 genes from the best-sampled 56 taxa. Maximum likelihood tree is shown as inferred using the LG + I + Γ model for each gene partition, and 100 bootstrap replicates. Filled blue circles represent 100% bootstrap support. The length of the matrix is 81,451 amino acids and overall matrix completeness is 89%. © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter Xenoturbella bocki 86 Isodiametra pulchra Eumecynostomum macrobursalium Childia submaculatum Convolutriloba macropyga Diopisthoporus gymnopharyngeus Diopisthoporus longitubus Hofstenia miamia Ascoparia sp. Meara stichopi Nemertoderma westbladi XENACOELOMORPHA Priapulus caudatus Halicryptus spinulosus Peripatopsis capensis Drosophila melanogaster Daphnia pulex Strigamia maritima 74 Ixodes scapularis Helobdella robusta Capitella teleta Pomatoceros lamarckii Cephalothrix hongkongiensis Lineus longissimus Phoronis psammophila Novocrania anomala Terebratalia transversa Hemithiris psittacea Leptochiton rugatus Crassostrea gigas Lottia gigantea Adineta ricciae Adineta vaga Brachionus calyciflorus 76 Lepidodermella squamata Macrodasys sp. Megadasys sp. 18 Macrostomum lignano Taenia pisiformes Schistosoma mansoni Schmidtea mediterranea Prostheceraeus vittatus Membranipora membranacea Loxosoma pectinaricola Barentsia gracilis Botryllus schlosseri Ciona intestinalis Petromyzon marinus Homo sapiens Gallus gallus Branchiostoma floridae Saccoglossus mereschkowskii Ptychodera bahamensis Schizocardium c.f. braziliense Cephalodiscus gracilis Dumetocrinus sp. Leptosynapta clarki Strongylocentrotus purpuratus Astrotomma agassizi Labidiaster annulatus Stomolophus meleagris Agalma elegans Craspedacusta sowerby CNIDARIA Nematostella vectensis Acropora digitifera Eunicella cavolinii Trichoplax adhaerens Aphrocallistes vastus Cliona varians Amphimedon queenslandica Leucosolenia complicata PORIFERA Sycon ciliatum Oscarella carmela Euplokamis dunlapae Pleurobrachia bachei CTENOPHORA Mnemiopsis leidyi Salpingoeca rosetta Monosiga brevicollis ECDYSOZOA SPIRALIA 79 97 DEUTEROSTOMIA 0.2 Extended Data Figure 2 | Maximum likelihood topology of metazoan relationships inferred from 881 genes and 77 taxa. Maximum likelihood tree is shown as inferred using the LG + I + Γ model for each gene partition, and 100 bootstrap replicates. Filled blue circles represent 100% bootstrap support. The length of the matrix is 337,954 amino acids and overall matrix completeness is 62%. © 2016 Macmillan Publishers Limited. All rights reserved Letter RESEARCH Xenoturbella bocki Priapulus caudatus Halicryptus spinulosus Peripatopsis capensis Strigamia maritima Daphnia pulex Drosophila melanogaster Ixodes scapularis Helobdella robusta Capitella teleta Pomatoceros lamarckii Phoronis psammophila Hemithiris psittacea Terebratalia transversa Novocrania anomala Cephalothrix hongkongiensis Lineus longissimus Leptochiton rugatus Crassostrea gigas Lottia gigantea Megadasys sp. Macrodasys sp. Brachionus calyciflorus Adineta vaga Adineta ricciae Lepidodermella squamata Macrostomum lignano Taenia pisiformes Schistosoma mansoni Schmidtea mediterranea 82 Prostheceraeus vittatus Membranipora membranacea Barentsia gracilis Loxosoma pectinaricola Dumetocrinus sp. Strongylocentrotus purpuratus Leptosynapta clarki Labidiaster annulatus Astrotomma agassizi Cephalodiscus gracilis Schizocardium c.f. braziliense Ptychodera bahamensis Saccoglossus mereschkowskii Ciona intestinalis Botryllus schlosseri Petromyzon marinus Homo sapiens Gallus gallus Branchiostoma floridae Stomolophus meleagris Craspedacusta sowerby Agalma elegans Eunicella cavolinii Acropora digitifera Nematostella vectensis Trichoplax adhaerens Sycon ciliatum Leucosolenia complicata Oscarella carmela Aphrocallistes vastus Amphimedon queenslandica Cliona varians Euplokamis dunlapae Mnemiopsis leidyi Pleurobrachia bachei Monosiga brevicollis Salpingoeca rosetta ECDYSOZOA 66 SPIRALIA 90 84 98 68 63 99 50 40 81 82 95 91 91 77 83 81 97 98 41 DEUTEROSTOMIA CNIDARIA 90 PORIFERA CTENOPHORA 0.2 Extended Data Figure 3 | Maximum likelihood topology of metazoan relationships inferred from 212 genes with Acoelomorpha removed. Maximum likelihood tree is shown as inferred using the LG + I + Γ model for each gene partition, and 100 bootstrap replicates. Filled blue circles represent 100% bootstrap support. The length of the matrix is 43,942 amino acids and overall matrix completeness is 70%. © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter 83 95 97 86 99 39 100 100 100 XENACOELOMORPHA 95 59 100 100 100 60 ECDYSOZOA 10 75 99 100 73 99 26 31 69 100 100 66 82 100 49 99 100 99 100 92 18 100 100 97 24 88 50 100 83 SPIRALIA 100 33 100 100 67 100 97 100 79 100 100 71 100 DEUTEROSTOMIA 100 31 98 7 81 100 100 100 100 98 91 83 100 100 100 100 100 100 Xenoturbella bocki Ascoparia sp. Nemertoderma westbladi Meara stichopi Sterreria sp. Hofstenia miamia Diopisthoporus longitubus Diopisthoporus gymnopharyngeus Isodiametra pulchra Eumecynostomum macrobursalium Convolutriloba macropyga Childia submaculatum Priapulus caudatus Halicryptus spinulosus Drosophila melanogaster Daphnia pulex Peripatopsis capensis Ixodes scapularis Strigamia maritima Membranipora membranacea Barentsia gracilis Loxosoma pectinaricola Helobdella robusta Capitella teleta Pomatoceros lamarckii Lineus longissimus Cephalothrix hongkongiensis Leptochiton rugatus Lottia gigantea Crassostrea gigas Phoronis psammophila Novocrania anomala Terebratalia transversa Hemithiris psittacea Brachionus calyciflorus Adineta vaga Adineta ricciae Macrodasys sp. Megadasys sp. Lepidodermella squamata Macrostomum lignano Prostheceraeus vittatus Schmidtea mediterranea Taenia pisiformes Schistosoma mansoni Branchiostoma floridae Ciona intestinalis Botryllus schlosseri Petromyzon marinus Homo sapiens Gallus gallus Cephalodiscus gracilis Saccoglossus mereschkowskii Ptychodera bahamensis Schizocardium c.f. braziliense Dumetocrinus sp. Strongylocentrotus purpuratus Leptosynapta clarki Labidiaster annulatus Astrotomma agassizi Trichoplax adhaerens Stomolophus meleagris Craspedacusta sowerby Agalma elegans CNIDARIA Eunicella cavolinii Acropora digitifera Nematostella vectensis Aphrocallistes vastus Amphimedon queenslandica Cliona varians PORIFERA Oscarella carmela Sycon ciliatum Leucosolenia complicata Euplokamis dunlapae Mnemiopsis leidyi CTENOPHORA Pleurobrachia bachei Salpingoeca rosetta Monosiga brevicollis 2.0 Extended Data Figure 4 | ASTRAL species tree, constructed from 212 input partial gene trees inferred in RAxML version 8.0.20. Nodal support values reflect the frequency of splits in trees constructed by ASTRAL from 100 bootstrap replicate gene trees. © 2016 Macmillan Publishers Limited. All rights reserved Letter RESEARCH Xenoturbella bocki Diopisthoporus longitubus Diopisthoporus gymnopharyngeus Hofstenia miamia Convolutriloba macropyga Childia submaculatum Eumecynostomum macrobursalium Isodiametra pulchra Meara stichopi Sterreria sp. Nemertoderma westbladi Ascoparia sp. XENACOELOMORPHA Halicryptus spinulosus Priapulus caudatus Drosophila melanogaster Daphnia pulex Ixodes scapularis Strigamia maritima Peripatopsis capensis Crassostrea gigas Lottia gigantea Leptochiton rugatus Hemithiris psittacea Terebratalia transversa Novocrania anomala Phoronis psammophila Cephalotrix hongkonggiensis Lineus longissimus Capitella teleta Pomatoceros lamarckii Helobdella robusta ECDYSOZOA SPIRALIA Taenia pisiformes Schistosoma mansoni Schmidtea mediterranea Prostheceraeus vittatus Macrostomum lignano Lepidodermella squamata Macrodasys sp. Megadasys sp. Brachionus calyciflorus Adineta ricciae Adineta vaga Barentsia gracilis Loxosoma pectinaricola Membranipora membranacea Labidiaster annulatus Astrotomma agassizi Strongylocentrotus purpuratus Leptosynapta clarki Dumetocrinus sp. Ptychodera bahamensis Schizocardium c.f. braziliense Saccoglossus merschkowskii Cephalodiscus gracilis Homo sapiens Gallus gallus Petromyzon marinus Botryllus schlosseri Ciona intestinalis Branchiostoma floridae Eunicella cavolinii Acropora digitifera Nematostella vectensis Agalma elegans Craspedacusta sowerby Stomolophus meleagris Trichoplax adhaerens Sycon ciliatum Leucosolenia complicata Oscarella carmela Amphimedon queenslandica Cliona varians Aphrocallistes vastus Euplokamis dunlapae Mnemiopsis leidyi Pleurobrachia bachei DEUTEROSTOMIA CNIDARIA PORIFERA Salpingoeca rosetta Monosiga brevicollis Extended Data Figure 5 | Bayesian inference topology of metazoan relationships inferred on the basis of 212 genes and 78 taxa. Results are shown from MrBayes analyses of four independent Metropolis-coupled CTENOPHORA chains run for 4,000,000 generations, with sampling every 500 generations. Amino-acid data were back-translated to nucleotides and analysed under an independent substitution model. © 2016 Macmillan Publishers Limited. All rights reserved RESEARCH Letter Extended Data Table 1 | Summary of data sets analysed in this study and support for monophyly of major groups Dataset description Number of OGs Number of Taxa AA positions % Missing Data Xenacoelomorpha Nephrozoa Bilateria HaMStR all taxa 212 78 44896 31 100/100/100 99/100/100 100/100/100 HaMStR best coverage taxa 336 56 81451 11 100 100 100 ProteinOrtho 881 77 337954 38 100 100 100 Remove Acoelomorpha 212 67 43942 30 N/A 81 100 Remove Xenoturbella 212 77 43510 31 (Acoelomorpha 100) 70 100 Remove Acoela 212 71 43451 31 100/100 100/1.0 100/100 Remove Nemertodermatida 212 74 45054 30 100 100 100 Remove Ctenophora 212 75 47011 30 100 100 100 Remove Cnidaria 212 72 44990 31 100 100 100 Remove Porifera 212 72 43829 31 100 100 100 Remove Placozoa 212 77 43940 31 100 100 100 Porifera only non-bilaterian Metazoa Remove non-metazoans 212 68 47115 30 100 100 100 212 76 43764 31 100 100 100 Reduce deuterostomes 210 74 46101 29 100 100 100 Taxa >80% gene occupancy only Taxa >90% gene occupancy only Remove taxa with LB score >13 Remove taxa with LB score >30 Genes with best LB scores 212 52 43868 16 100 99 100 212 40 42840 11 100 99 100 212 59 43247 30 100 98 100 212 73 44260 30 100 100 100 106 78 22295 30 100 71 100 Genes with poor LB scores 106 78 22601 32 100 99 100 Genes with lowest saturation Genes with highest saturation Only non-ribosomal protein genes Ribosomal protein genes, LG all partitions BMGE trimming 106 78 23414 29 100 95 100 106 78 21482 34 100 100 100 207 78 44715 32 100 100 100 53 78 9010 19 non-monophyletic Merged 78 33323 34 100 non-monophyletic 100 88 100 Bootstrap support values given from RAxML analyses inferred with the LG + I + Γ model from 100 rapid bootstrap replicates. Bayesian posterior probabilities are listed from MrBayes analyses inferred under an independent substitution model using a back-translated nucleotide data set derived from our amino-acid alignment, and PhyloBayes analyses under the CAT + GTR + Γ model. ! © 2016 Macmillan Publishers Limited.