Xenacoelomorpha is the sister group to Nephrozoa

Abstract

The position of Xenacoelomorpha in the tree of life remains a major unresolved question in the study of deep animal relationships. 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) or is a clade inside Deuterostomia. 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 long-branch 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.

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4 FEBRUARY 2016 | VOL 530 | NATURE | 89
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 long-
branch 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 lin-
eage7,8. When nucleotide sequence data became available, Acoela were
placed as the sister group of Nephrozoa9. Nemertodermatida were orig
-
inally classified within Acoela, but were soon recognized as a separate
clade on morphological grounds
10
. 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 bilaterians
15
. The first molecular study suggested Xenoturbella
to be closely related to molluscs
16
, whereas other analyses proposed
a deuterostome affiliation
17,18
. Recent analyses of molecular data reu-
nited Xenoturbella with acoels and nemertodermatids
2–4
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 Bilateria
19
(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 mitochon-
drial 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 acoelo-
morphs 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 parsimony
4,22
and under Bayesian inference
22
.
Phylogenomic analyses recovering xenacoelomorph taxa within
Deuterostomia show branching patterns that differ significantly
1Naturhistoriska 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.
Xenoturbella
Ambulacraria
Chordata
Protostomia
Bilateria
Deuterostomia
Acoelomorpha
Bilateria
Xenacoelomorpha
Ambulacraria
Chordata
Protostomia
Nephrozoa
Deuterostomia
Xenacoelomorpha
Ambulacraria
Chordata
Protostomia
Bilateria
Deuterostomia
Xenambulacraria
a
bc
de
Xenoturbella
Xenacoelomorpha
Bilateria Xenacoelomorpha
Ambulacraria
Chordata
Protostomia
Deuterostomia
Nephrozoa
Nemertodermatida Acoela
Xenambulacraria
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).
© 2016 Macmillan Publishers Limited. All rights reserved

Letter
reSeArCH
90 | NATURE | VOL 530 | 4 FEBRUARY 2016
between alternative models of evolution
4
. Conflicting results in stud-
ies that used the same expressed sequence tag data for xenacoelo-
morphs2,4 suggest some degree of model misspecification, missing
data generating positively misleading signal, or long-branch attrac-
tion (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 spe-
cies, 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, Diopisthoporidae23. 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 morpho-
logical 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 ami-
no-acid positions, and 31% missing data (Extended Data Table 1).
0.2
Terebratalia transversa
Capitella teleta
Novocrania anomala
Oscarella carmela
Macrodasys sp.
Crassostrea gigas
Botryllus schlosseri
Loxosoma pectinaricola
Xenoturbella bocki
Astrotomma agassizi
Sterreria sp.
Pomatoceros lamarckii
Brachionus calyciflorus
Dumetocrinus sp.
Euplokamis dunlapae
Membranipora membranacea
Isodiametra pulchra
Amphimedon queenslandica
Ciona intestinalis
Lottia gigantea
Childia submaculatum
Salpingoeca rosetta
Lineus longissimus
Strongylocentrotus purpuratus
Agalma elegans
Trichoplax adhaerens
Diopisthoporus gymnopharyngeus
Megadasys sp.
Ptychodera bahamensis
Acropora digitifera
Adineta vaga
Peripatopsis capensis
Daphnia pulex
Convolutriloba macropyga
Adineta ricciae
Phoronis psammophila
Schizocardium braziliense
Schistosoma mansoni
Helobdella robusta
Prostheceraeus vittatus
Pleurobrachia bachei
Eunicella cavolinii
Leptochiton rugatus
Labidiaster annulatus
Barentsia gracilis
Halicryptus spinulosus
Gallus gallus
Eumecynostomum macrobursalium
Strigamia maritima
Nematostella vectensis
Ixodes scapularis
Cliona varians
Mnemiopsis leidyi
Diopisthoporus longitubus
Meara stichopi
Craspedacusta sowerby
Leucosolenia complicata
Monosiga brevicollis
Priapulus caudatus
Hemithiris psittacea
Ascoparia sp.
Cephalothrix hongkongiensis
Hofstenia miamia
Saccoglossus mereschkowskii
Aphrocallistes vastus
Taenia pisiformes
Petromyzon marinus
Sycon ciliatum
Lepidodermella squamata
Nemertoderma westbladi
Schmidtea mediterranea
Drosophila melanogaster
Cephalodiscus gracilis
Stomolophus meleagris
Homo sapiens
Leptosynapta clarki
Branchiostoma floridae
Macrostomum lignano
95/99/95
86/76/86
70/
74/
70
66/78/66
92/96/92
98/98/98
77/52/77
67/--/67
95/99/95
55/53/55
89/97/89
63/76/63
67/--/67
78/85/78
99/
100/
99
96/100/96
71/60/71
39/43/39
47/65/47
87/83/87
Xenacoelomorpha
Nemertodermatida
Platyhelminthes
Lophophorata
(Phoronida + Brachiopoda)
Mollusca
Spiralia
Rotifera
Gastrotricha
Entoprocta
Bryozoa
Onychophora
Arthropoda
Priapulida
Placozoa
Choanoagellata
Ambulacraria
(Echinodermata
+Hemichordata)
BS support =100% all models, ProtTest/LG4X/LG
Acoela
Ecdysozoa
Nephrozoa
Bilateria
Deuterostomia
Cnidaria
Annelida
Ctenophora
Porifera
Chordata
Nemertea
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.
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Letter reSeArCH
4 FEBRUARY 2016 | VOL 530 | NATURE | 91
Sequences were taken entirely from Illumina transcriptomes or pre-
dicted 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 phy-
logenomic 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 best-
fitting model for each individual gene partition, or the LG model, or
the LG4X model
24
over each independent partition. The LG4X model
is composed of four substitution matrixes designed to improve mod-
elling of site heterogeneity24. Bayesian analyses were conducted with
the site-heterogeneous CAT + GTR + Γ model and GTR + Γ. To fur-
ther 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
0.3
Leptochiton rugatus
Oscarella carmela
Botryllus schlosseri
Strigamia maritima
Astrotomma agassizi
Eumecynostomum macrobursalium
Branchiostoma floridae
Hofstenia miamia
Adineta ricciae
Stomolophus meleagris
Drosophila melanogaster
Leptosynapta clarki
Priapulus caudatus
Meara stichopi
Schistosoma mansoni
Homo sapiens
Cephalothrix hongkongiensis
Strongylocentrotus purpuratus
Gallus gallus
Acropora digitifera
Convolutriloba macropyga
Eunicella cavolinii
Hemithiris psittacea
Phoronis psammophila
Lineus longissimus
Diopisthoporus gymnopharyngeus
Membranipora membranacea
Megadasys sp.
Taenia pisiformes
Peripatopsis capensis
Sycon ciliatum
Macrostomum lignano
Nematostella vectensis
Childia submaculatum
Diopisthoporus longitubus
Saccoglossus mereschkowskii
Terebratalia transversa
Macrodasys sp.
Mnemiopsis leidyi
Dumetocrinus sp.
Ascoparia sp.
Aphrocallistes vastus
Pleurobrachia bachei
Leucosolenia complicata
Amphimedon queenslandica
Monosiga brevicollis
Capitella teleta
Halicryptus spinulosus
Adineta vaga
Petromyzon marinus
Isodiametra pulchra
Labidiaster annulatus
Prostheceraeus vittatus
Lepidodermella squamata
Daphnia pulex
Barentsia gracilis
Craspedacusta sowerby
Schmidtea mediterranea
Nemertoderma westbladi
Cliona varians
Agalma elegans
Euplokamis dunlapae
Sterreria sp.
Ciona intestinalis
Ptychodera bahamensis
Xenoturbella bocki
Helobdella robusta
Pomatoceros lamarckii
Salpingoeca rosetta
Cephalodiscus gracilis
Brachionus calyciflorus
Ixodes scapularis
Schizocardium c.f. braziliense
Trichoplax adhaerens
Lottia gigantea
Novocrania anomala
Loxosoma pectinaricola
Crassostrea gigas
0.94
0.78
0.99
0.88
0.83
0.99
0.51
Xenacoelomorpha
Spiralia
Ecdysozoa
Deuterostomia
Cnidaria
Ctenophora
Porifera
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.
© 2016 Macmillan Publishers Limited. All rights reserved

Letter
reSeArCH
92 | NATURE | VOL 530 | 4 FEBRUARY 2016
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 arte-
facts, and genes or positions that were more saturated. Using our stand-
ard pipeline, for the best-sampled 56 taxa, we also generated a data
set with 336 orthologous groups, 81,451 amino acids, and 11% miss-
ing 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 amino-
acid 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 likeli-
hood 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 posi-
tion 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 meta-
zoan 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 larg-
est 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.
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
organs
13
. Nemertodermatida also have an epithelial gut, but the mouth
appears to be a transient structure
10
. 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 con-
siderable morphological variation: acoels have no intestinal lumen
although a mouth opening and sometimes a pharynx is present
23
. The
nervous system is highly variable, there are one or two gonopores, and
often accessory reproductive organs
23
. The morphological evolution
that occurred within Xenacoelomorpha provides an interesting par-
allel case to Nephrozoa.
The sister group relationship between Xenacoelomorpha and
Nephrozoa allows us to infer the order in which bilaterian features
were evolved
12,29
. 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.
Annelida
Nemertodermatida
Brachiopoda
Cnidaria
Placozoa
Platyhelminthes
Entoprocta
Xenoturbella
Bryozoa
Ctenophora
Arthropoda
Choanoagellata
Onychophora
Porifera
Nemertea
Phoronida
Mollusca
Priapulida
Ambulacraria
Rotifera
Acoela
Gastrotricha
Chordata
Xenacoelomorpha
Ecdysozoa
Trochozoa
Spiralia
Protostomia
Deuterostomia
Nephrozoa
Bilateria
Planulozoa
Metazoa
Parahoxozoa
= Maximal support in analyses
of 212, 336, and 881 genes
Bilateria
Xenacoelomorpha
Deuterostomia
Protostomia
Cnidaria
Mesoderm
Nephridia
Bilaterality
Sac-like
epithelial gut
Nerve-net
Nephrozoa
a
b
>70% >99%
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.
© 2016 Macmillan Publishers Limited. All rights reserved

Letter reSeArCH
4 FEBRUARY 2016 | VOL 530 | NATURE | 93
Online Content Methods, along with any additional Extended Data display items and
Source Data, are available in the online version of the paper; references unique to
these sections appear only in the online paper.
Received 19 September; accepted 7 December 2015.
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within the deuterostomes. BMC Evol. Biol. 9, 107 (2009).
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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).
22. Thomson, R. C., Plachetzki, D. C., Mahler, D. L. & Moore, B. R. A critical appraisal
of the use of microRNA data in phylogenetics. Proc. Natl Acad. Sci. USA 111,
E3659–E3668 (2014).
23. Jondelius, U., Wallberg, A., Hooge, M. & Raikova, O. I. How the worm got its
pharynx: phylogeny, classication and Bayesian assessment of character
evolution in Acoela. Syst. Biol. 60, 845–871 (2011).
24. Le, S. Q., Dang, C. C. & Gascuel, O. Modeling protein evolution with several
amino acid replacement matrices depending on site rates. Mol. Biol. Evol. 29,
2921–2936 (2012).
25. Dunn, C. W. et al. Broad phylogenomic sampling improves resolution of the
animal tree of life. Nature 452, 745–749 (2008).
26. Mirarab, S. et al. ASTRAL: genome-scale coalescent-based species tree
estimation. Bioinformatics 30, i541–i548 (2014).
27. Bleidorn, C. et al. On the phylogenetic position of Myzostomida: can 77 genes
get it wrong? BMC Evol. Biol. 9, 150 (2009).
28. Whelan, N. V., Kocot, K. M., Moroz, L. L. & Halanych, K. M. 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).
© 2016 Macmillan Publishers Limited. All rights reserved

Letter
reSeArCH
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 metazo-
ans (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 avail
-
able 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 deter-
mine 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 ProteinOrtho
33
. 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 charac-
ters 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 gener-
ated 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 align-
ment 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 ort-
hologous group alignments were realigned using MAFFT following the removal of
selected taxa. TreSpEx
41
was used to assess potential sources of misleading signal,
including standard deviation of branch-length heterogeneity (LB) and satura-
tion. 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 ort-
hologous 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-MPI
45
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 nucleo-
tides 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 sec-
ond 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, mod-
elling rate variation across sites using a discrete gamma distribution (four catego-
ries) with a proportion of invariable sites (‘lset rates = invgamma’), and nucleotide
substitutions with independent stationary state frequencies and a reversible-
jump approach to the partitioning of exchangeability rates (‘lset nst = mixed’).
We also uncoupled the partition rates (‘prset ratepr = variable’). All other set-
tings 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 genera-
tions, 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 ASTRAL
26
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: prole 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 identies
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 maximum-
likelihood 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-t models of protein evolution. Bioinformatics 27, 1164–1165 (2011).
45. Lartillot, N., Rodrigue, N., Stubbs, D. & Richer, J. PhyloBayes MPI: phylogenetic
reconstruction with innite mixtures of proles in a parallel environment.
Syst. Biol. 62, 611–615 (2013).
46. Ronquist, F. et al. MrBayes 3.2: ecient 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
0.2
Adineta ricciae
Gallus gallus
Macrostomum lignano
Diopisthoporus longitubus
Hofstenia miamia
Homo sapiens
Dumetocrinus sp.
Childia submaculatum
Drosophila melanogaster
Sycon ciliatum
Adineta vaga
Convolutriloba macropyga
Branchiostoma floridae
Ptychodera bahamensis
Brachionus calyciflorus
Strigamia maritima
Stomolophus meleagris
Xenoturbella bocki
Saccoglossus mereschkowskii
Membranipora membranacea
Botryllus schlosseri
Daphnia pulex
Agalma elegans
Craspedacusta sowerby
Nemertoderma westbladi
Ciona intestinalis
Halicryptus spinulosus
Novocrania anomala
Crassostrea gigas
Amphimedon queenslandica
Isodiametra pulchra
Priapulus caudatus
Oscarella carmela
Phoronis psammophila
Lepidodermella squamata
Salpingoeca rosetta
Leptochiton rugatus
Monosiga brevicollis
Lottia gigantea
Lineus longissimus
Macrodasys sp.
Meara stichopi
Cliona varians
Helobdella robusta
Nematostella vectensis
Terebratalia transversa
Aphrocallistes vastus
Prostheceraeus vittatus
Trichoplax adhaerens
Hemithiris psittacea
Mnemiopsis leidyi
Capitella teleta
Strongylocentrotus purpuratus
Ixodes scapularis
Schistosoma mansoni
Leucosolenia complicata
72
90
56
56
98
80
96
64
44
98
44
XENACOELOMORPHA
ECDYSOZOA
DEUTEROSTOMIA
CNIDARIA
CTENOPHORA
PORIFERA
SPIRALIA
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

Letter
reSeArCH
0.2
Ixodes scapularis
Ciona intestinalis
Amphimedon queenslandica
Adineta vaga
Schizocardium c.f. braziliense
Cliona varians
Taenia pisiformes
Mnemiopsis leidyi
Lineus longissimus
Ascoparia sp.
Phoronis psammophila
Membranipora membranacea
Childia submaculatum
Salpingoeca rosetta
Hemithiris psittacea
Monosiga brevicollis
Adineta ricciae
Macrostomum lignano
Priapulus caudatus
Agalma elegans
Brachionus calyciflorus
Botryllus schlosseri
Terebratalia transversa
Megadasys sp.
Cephalothrix hongkongiensis
Diopisthoporus longitubus
Lepidodermella squamata
Halicryptus spinulosus
Leptosynapta clarki
Homo sapiens
Euplokamis dunlapae
Nemertoderma westbladi
Diopisthoporus gymnopharyngeus
Nematostella vectensis
Trichoplax adhaerens
Helobdella robusta
Macrodasys sp.
Schmidtea mediterranea
Barentsia gracilis
Drosophila melanogaster
Convolutriloba macropyga
Peripatopsis capensis
Ptychodera bahamensis
Petromyzon marinus
Saccoglossus mereschkowskii
Craspedacusta sowerby
Isodiametra pulchra
Pomatoceros lamarckii
Cephalodiscus gracilis
Strigamia maritima
Branchiostoma floridae
Schistosoma mansoni
Gallus gallus
Astrotomma agassizi
Daphnia pulex
Capitella teleta
Leptochiton rugatus
Acropora digitifera
Meara stichopi
Lottia gigantea
Novocrania anomala
Stomolophus meleagris
Leucosolenia complicata
Hofstenia miamia
Eunicella cavolinii
Pleurobrachia bachei
Crassostrea gigas
Dumetocrinus sp.
Prostheceraeus vittatus
Loxosoma pectinaricola
Xenoturbella bocki
Labidiaster annulatus
Aphrocallistes vastus
Sycon ciliatum
Eumecynostomum macrobursalium
Strongylocentrotus purpuratus
Oscarella carmela
74
97
18
76
86
79
XENACOELOMORPHA
SPIRALIA
ECDYSOZOA
DEUTEROSTOMIA
CNIDARIA
CTENOPHORA
PORIFERA
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
0.2
Priapulus caudatus
Trichoplax adhaerens
Euplokamis dunlapae
Sycon ciliatum
Leptochiton rugatus
Loxosoma pectinaricola
Saccoglossus mereschkowskii
Leptosynapta clarki
Ptychodera bahamensis
Adineta vaga
Cephalodiscus gracilis
Lepidodermella squamata
Mnemiopsis leidyi
Labidiaster annulatus
Leucosolenia complicata
Amphimedon queenslandica
Strigamia maritima
Megadasys sp.
Terebratalia transversa
Macrodasys sp.
Macrostomum lignano
Astrotomma agassizi
Membranipora membranacea
Gallus gallus
Branchiostoma floridae
Hemithiris psittacea
Halicryptus spinulosus
Taenia pisiformes
Daphnia pulex
Agalma elegans
Phoronis psammophila
Novocrania anomala
Botryllus schlosseri
Crassostrea gigas
Schmidtea mediterranea
Monosiga brevicollis
Barentsia gracilis
Stomolophus meleagris
Adineta ricciae
Schizocardium c.f. braziliense
Aphrocallistes vastus
Pleurobrachia bachei
Xenoturbella bocki
Peripatopsis capensis
Lottia gigantea
Cliona varians
Pomatoceros lamarckii
Cephalothrix hongkongiensis
Nematostella vectensis
Petromyzon marinus
Craspedacusta sowerby
Strongylocentrotus purpuratus
Helobdella robusta
Ciona intestinalis
Oscarella carmela
Prostheceraeus vittatus
Dumetocrinus sp.
Schistosoma mansoni
Drosophila melanogaster
Capitella teleta
Ixodes scapularis
Brachionus calyciflorus
Acropora digitifera
Eunicella cavolinii
Salpingoeca rosetta
Lineus longissimus
Homo sapiens
66
95
84
98
81
81
77
82
68
40
91
98
83
90
91
99
41
97
82
63
50
90
SPIRALIA
ECDYSOZOA
DEUTEROSTOMIA
CNIDARIA
CTENOPHORA
PORIFERA
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

Letter
reSeArCH
2.0
Childia submaculatum
Petromyzon marinus
Stomolophus meleagris
Prostheceraeus vittatus
Agalma elegans
Pomatoceros lamarckii
Drosophila melanogaster
Acropora digitifera
Strigamia maritima
Macrodasys sp.
Phoronis psammophila
Amphimedon queenslandica
Diopisthoporus gymnopharyngeus
Xenoturbella bocki
Loxosoma pectinaricola
Astrotomma agassizi
Mnemiopsis leidyi
Nemertoderma westbladi
Salpingoeca rosetta
Peripatopsis capensis
Adineta ricciae
Euplokamis dunlapae
Gallus gallus
Leptosynapta clarki
Isodiametra pulchra
Ixodes scapularis
Terebratalia transversa
Macrostomum lignano
Adineta vaga
Branchiostoma floridae
Hemithiris psittacea
Schmidtea mediterranea
Strongylocentrotus purpuratus
Meara stichopi
Sterreria sp.
Priapulus caudatus
Lepidodermella squamata
Leucosolenia complicata
Labidiaster annulatus
Ascoparia sp.
Cephalothrix hongkongiensis
Ciona intestinalis
Trichoplax adhaerens
Oscarella carmela
Lineus longissimus
Diopisthoporus longitubus
Helobdella robusta
Novocrania anomala
Aphrocallistes vastus
Cliona varians
Leptochiton rugatus
Pleurobrachia bachei
Brachionus calyciflorus
Eumecynostomum macrobursalium
Schistosoma mansoni
Megadasys sp.
Dumetocrinus sp.
Saccoglossus mereschkowskii
Schizocardium c.f. braziliense
Hofstenia miamia
Capitella teleta
Halicryptus spinulosus
Monosiga brevicollis
Lottia gigantea
Eunicella cavolinii
Taenia pisiformes
Nematostella vectensis
Crassostrea gigas
Membranipora membranacea
Ptychodera bahamensis
Barentsia gracilis
Sycon ciliatum
Homo sapiens
Cephalodiscus gracilis
Daphnia pulex
Craspedacusta sowerby
Convolutriloba macropyga
Botryllus schlosseri
100
100
81
100
86
31
100
100
100
100
92
100
95
100
100
75
100
98
100
98
97
99
7
31
100
99
18
71
100
66
100
100
69
24
33
100
39
26
100
100
79
50
67
83
83
88
100
100
100
97
82
91
95
99
100
99
100
83
97
100
59
100
100
100
100
49
100
100
73
100
60
100
100
99
10
ECDYSOZOA
SPIRALIA
XENACOELOMORPHA
DEUTEROSTOMIA
CTENOPHORA
PORIFER
A
CNIDARIA
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
XENACOELOMORPHA
SPIRALIA
ECDYSOZOA
DEUTEROSTOMIA
CNIDARIA
CTENOPHORA
PORIFERA
Xenoturbella bocki
Diopisthoporus longitubus
Diopisthoporus gymnopharyngeus
Hofstenia miamia
Convolutriloba macropyga
Childia submaculatum
Eumecynostomum macrobursalium
Isodiametra pulchra
Meara stichopi
Sterreria sp.
Nemertoderma westbladi
Ascoparia sp.
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
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
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
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

Letter
reSeArCH
Extended Data Table 1 | Summary of data sets analysed in this study and support for monophyly of major groups
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.
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
212 68 47115 30 100 100 100
Remove non-metazoans 212 76 43764 31 100 100 100
Reduce deuterostomes 210 74 46101 29 100 100 100
Taxa >80% gene occupancy
only
212 52 43868 16 100 99 100
Taxa >90% gene occupancy
only
212 40 42840 11 100 99 100
Remove taxa with LB score
>13
212 59 43247 30 100 98 100
Remove taxa with LB score
>30
212 73 44260 30 100 100 100
Genes with best LB scores 106 78 22295 30 100 71 100
Genes with poor LB scores 106 78 22601 32 100 99 100
Genes with lowest
saturation
106 78 23414 29 100 95 100
Genes with highest
saturation
106 78 21482 34 100 100 100
Only non-ribosomal protein
genes
207 78 44715 32 100 100 100
Ribosomal protein genes,
LG all partitions
53 78 9010 19 non-monophyletic non-monophyletic 88
BMGE trimming Merged 78 33323 34 100 100 100
!
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