Back in time: a new sys
la
z, Jordi Paps and Marta Riutort
gia
celo
nism
a si
ce
Pla
o
an
eq
es
chi
ap
th
nu
recent gene expression data suggest that cnidaria
common bilaterian ancestor has to be put back in t
sc
rto
s
o
(
1. INT
When
molecu
netic r
they b
increas
genetic
increas
Cavali
and se
clock-l
Paulin
a perfe
would
both
igher
iption
other
ity in
ith or
ctions
rates
ything
otein,
Nei &
long-
ncient
urred
ls and
ctory
ortant
ateral
triploblastic, which means the presence of a third
middle layer or mesoderm, from which most organs1098/rstb.2007.2238 or via http://journals.royalsociety.org.
One contribution of 17 to a Discussion Meeting Issue ‘Evolution of
the animals: a Linnean tercentenary celebration’.used to guide an understanding of the evolution of
morphological characters. 2. THE BILATERIA: BASIC FEATURES AND TWOQUESTIONS
Bilaterians include all Metazoa with bilateral symmetry
either in the adult stage or, in those cases where
bilateral symmetry turned to radial symmetry (e.g.
echinoderms), in the larval stage. All bilaterians are
Electronic supplementary material is available at http://dx.doi.org/10.
*Author for correspondence (jbaguna@ub.edu).main cladogenetic events. Morphological characters
and new genes appearing at each node could then behe time will come I believe, though I shall not live to
ee it, when we shall have fairly true genealogical trees
f each great kingdom of nature.
Charles Darwin in a letter to Thomas Huxley, 1857)
RODUCTION
in the last third of the twentieth century
lar taxonomists aimed to establish the phyloge-
elationships of all organisms (the Tree of Life),
egan with the following two premises: first, the
e in morphological complexity along the phylo-
tree should run parallel to and be based on an
e in genomic complexity (e.g. number of genes;
er-Smith 1985; reviewed in Hahn & Wray 2002)
cond, genes and proteins should have a constant,
ike rate of change over time (Zuckerkandl &
g 1965; Kimura & Ohta 1973). Therefore, under
ct molecular clock, protein and DNA sequences
result in a complete Tree of Life delineating its
Some clues emerging from molecular biolog
developmental genetics in the early 1990s proved
premises to be flawed. First, lower and h
organisms encode very similar families of transcr
factors and signal transduction molecules. In
words, variation in morphological complex
metazoan evolution is probably correlated w
caused by the variation in the amount of intera
of a more or less similar set of genes. Second, the
of change in genes and proteins proved to be an
but clock-like and vary according to the gene, pr
lineage, site and period studied (Easteal 1985;
Kumar 2000).
One main consequence of these changes is the
lasting difficulties in resolving the so-called a
radiations, that is, cladogenetic events which occ
a long time ago and for which morphology, fossi
molecules have, so far, not provided satisfa
answers. Here, we address arguably the most imp
conundrum: the origin and radiation of bil
animals (the Bilateria).T y andthe form of a planuloid animal. A new systematic
is suggested and its main implications discussed.
Keywords: Bilateria; Cnidaria; Acoela; Nemethe Bi
Jaume Bagun˜a`*, Pere Martine
Departament de Gene`tica, Facultat de Biolo
08028 Bar
Conventional wisdom suggests that bilateral orga
than bilaterally, symmetrical and, therefore, had
hypotheses on how this transformation took pla
planula larva of extant cnidarians or the acoel
rather complex organism bearing several or m
(archicoelomate theory). We report phylogenetic
(ribosomal, nuclear and expressed sequence tag s
microRNA sets) markers. The phylogenetic tre
nemertodermatid flatworms as the earliest bran
some acoelomate and pseudocoelomate clades
deuterostomes. These results strengthen the view
docoelomate, benthic organisms derived from pla1, Universitat de Barcelona, Diagonal 645,
na, Spain
s arose from ancestors that were radially, rather
ngle body axis and no mesoderm. The two main
consider either a simple organism akin to the
tyhelminthes (planuloid–acoeloid theory), or a
st features of advanced coelomate bilaterians
alyses of bilaterian metazoans using quantitative
uences) and qualitative (HOX cluster genes and
obtained corroborate the position of acoel and
ng extant members of the Bilateria. Moreover,
pear as early branching lophotrochozoans and
at stem bilaterians were small, acoelomate/pseu-
loid-like organisms. Because morphological and
ns are actually bilateral, the origin of the last
ime earlier than the cnidarian–bilaterian split in
heme for the Bilateria that includes the Cnidaria
dermatida; molecular phylogeny; microRNAtematic proposal for
teria
Phil. Trans. R. Soc. B
doi:10.1098/rstb.2007.2238
Published onlineThis journal is q 2008 The Royal Society

2 J. Bagun˜a` et al. Phylogeny of the Bilateriastem(a)
(b)
a b c
a b c
d
P/D LCA
LCBA
e f
g h i j k
j′ k′
j′′k′′m
l
d e f g h i j k
crown
LCBA
( =P/D LCA)
Bilateria
Radiata
Bilateria
Radiata
Figure 1. Coincident changes in the branch leading to the
LCBA node under (a) the complex Urbilateria hypothesis
(archicoelomate theory; Remane 1963; Kimmell 1996;
Adoutte et al. 2000) and (b) the simple Urbilateria hypothesis
(planuloid–acoeloid theory; Hyman 1951; Salvini-Plawen
1978). Note that in the complex Urbilateria scenario,
characters (a–k) clump at the LCBA node that by definition
corresponds to the last common ancestor of protostomes and
deuterostomes (P/D LCA). Under the simple Urbilateriaform; so, true organs arise only in the triploblasts.
Finally, many bilateral animals show a concentration of
sensory structures and nerve cells at the anterior end of
the body (e.g. cephalization). These features are widely
considered basic apomorphies for the Bilateria.
However, two questions remain. First, did the first
bilaterian bear just this basic set of characters or, as
some theories and hypotheses suggest (see below), did
they also feature other characters (e.g. true brain,
through gut, excretory system, body cavities (coelom),
segments and even appendages and simple hearts and
eyes) making them rather complex organisms? Second,
do some non-bilaterian clades, traditionally considered
radially or biradially symmetric (e.g. cnidarians),
exhibit bilateral features and, hence, should they be
considered true bilaterians?
The scores of theories advanced since Haeckel’s
gastraea theory on the nature of the first bilaterian (for
recent updates see Willmer (1990) and Holland
(2003)) could be separated into two main groups.
The archicoelomate theories contemplate basic bilater-
ian traits such as bilaterality (hence a D–V axis) and
mesoderm appearing concurrently with advanced
characters such as coelom and segments. Therefore,
the first bilaterians were segmented and coelomate and
derived from radially symmetric, non-segmented,
acoelomate cnidarians, either under larval or adult
hypothesis, new clades intercalate and separate the LCBA
from the P/D LCA helping to distribute character changes
(a–m) across a series of stem branches and to polarize them.
Under this scenario, the LCBA is morphologically simpler
than the P/D LCA. Note that characters j and k could be
either monophyletic ( j,k) or di- or polyphyletic (j 0, j 00, k 0, k 00).
l and m represent protostome- and deuterostome-specific
characters. Grey ovals indicate the stem branches where key
innovations (new characters) appeared.
Phil. Trans. R. Soc. Bappearance (Remane 1963; Holland 2003). Under this
hypothesis, the last common bilaterian ancestor
(LCBA) appears as a rather complex organism
(dubbed complex Urbilateria; De Robertis & Sasai
1996; Kimmell 1996; Carroll et al. 2001) and is defined
as the last common ancestor of Protostomia and
Deuterostomia (hence, the P/D LCA; for a clarifying
terminology, see Valentine (2006)). The alternative
group of theories feature a more gradual scenario
starting from sexually reproducing pelagic organisms
(protoplanula or archiplanula), akin to present-day
cnidarian planula larvae, already exhibiting some
bilateral symmetry (see Salvini-Plawen (1978) for a
thorough review). Under this scenario, the LCBA was
a morphologically simple organism and the P/D LCA
would be relegated to an internal node within the
Bilateria. From this simple LCBA originated the
cnidarian polyps, which settled on the substratum, as
well as a stock of acoelomate, non-segmented, early
bilaterians vaguely similar to present-day acoel and
nemertodermatid flatworms (planuloid–acoeloid
theory). From the latter stock, other acoelomates as
well as pseudocoelomate and coelomate, segmented
and non-segmented protostomes and deuterostomes
gradually evolved.
In terms of character changes necessary between
ancestors and descendants, the phylogenetic conse-
quences of these conflicting scenarios are very different.
Under the archicoelomate scenario, the number
of coincident characters present at the LCBA (ZP/D
LCA) node is large (figure 1a). This makes it difficult to
place them into any temporal order along the stem
leading to the LCBA. It also implies either a large
number of extinctions of intermediary taxa (stem
ancestors) or a wholesale correlated transformation
from one life form (radial) to another (bilateral). In
contrast, the planuloid–acoeloid scenario posits a
reduced number of characters at the stem leading to
the LCBA and features fewer and simpler stem
ancestors, a simple LCBA and a later origin for the
P/D LCA (figure 1b). Hopefully, and under both
scenarios, phylogenetic advances may uncover fossil or
extant clades that break coincident character changes
at the stem. The intercalation of these new clades will
distribute inferred character changes across a series of
branches instead of having them solely at the LCBA
node (Donoghue 2005; Valentine 2006).
As regards whether clades outside the Bilateria do
exhibit bilateral symmetry, recent genomic and gene
expression analyses have shown that besides genes
involved in A–P polarity, gastrulation, endodermal and
germ cell specifications, cnidarian anthozoans have
numerous orthologues of bilaterian gene families
previously thought to be absent in ‘radial’ organisms
(for specific references see Finnerty et al. 2004;
Martindale et al. 2004; Martindale 2005; Matus et al.
2006a; Rentzsch et al. 2006). Importantly, the presence
and expression in cnidarians of many of the genes
involved in D–V patterning in bilaterians match ideas
(going back to Stephenson (1926) and held by Hyman
(1951) and Salvini-Plawen (1978)) of a second
or directive axis in cnidarians (specifically in anthozo-
ans), perpendicular to the oral–aboral (O–AB) axis
(Finnerty et al. 2004). Were it so, cnidarians and

homologizing morphological structures on the basis of
gene expression alone (Abouheif 1997; Wagner 2007).
2006). However, besides stochastic errors and short
Phylogeny of the Bilateria J. Bagun˜a` et al. 3Such difficulties make the use of molecular char-
acters for reconstructing a backbone tree of the
Metazoa and the Bilateria an attractive option. There-
after, morphological and gene expression charactersbilaterians might have evolved from an already bilateral
ancestor, putting the origin of the bilaterians even
further back in time.
3. CURRENT APPROACHES TO UNRAVEL THE
ORIGIN AND EVOLUTION OF BILATERIANS
To establish whether the first bilaterians had the basic
or an expanded set of embryological/morphological key
characters or novelties (see above), two main
approaches are currently used: (i) molecular phyloge-
nies to sample taxa as close as possible on either side of
the origin of evolutionary novelties and (ii) comparing
the expression patterns of homologous genes related to
these novelties among bilaterians and non-bilaterians
as a criterion of homology of the corresponding
anatomical structures.
Building molecular phylogenetic trees under rigor-
ous phylogenetic inference methods aims to identify
potential earliest branching bilaterians bearing novel-
ties (e.g. symmetry, mesoderm, through gut, nephridia,
coelom, segments, etc.), derived from ancestors that
did not possess such features. Extant ‘non-bilaterian’ or
‘pre-bilaterian’ metazoan groups must also be searched
for to be used as appropriate outgroups. As Raff (2000)
states, phylogeny provides three important kinds of
information: (i) it can determine the direction in which
developmental features evolve, (ii) it allows evolution-
ary rates to be inferred, and (iii) it allows homology
statements to be formulated or, conversely, tested.
Information of type (i) and (iii) is particularly important
as it helps to determine the ‘true’ groups before and
after a morphological novelty and so avoid mistaken
comparisons of gene expression patterns in non-
homologous features (see below).
The rationale behind comparing expression patterns
of developmental control genes between closely or
distantly related taxa is that if in two different species
orthologous genes are expressed in a similar position,
these areas or regions are considered homologous, even
across phyla, and should have been present in their last
common ancestor. However, attempting to infer
structural homology from molecular expression is
fraught with difficulties (Abouheif 1997). Some of the
genes tested (namely the HOX and some D–V genes;
Arendt & Nu¨bler-Jung 1994; De Robertis & Sasai
1996) are good examples of homologous genes used
across phyla in homologous patterning mechanisms
that result in rather different structures, i.e. HOX genes
patterning the A–P axis in arthropods and chordates.
Other sets of genes whose expression in embryos was
used to deduce homology of structures across phyla
(i.e. DLL/distal-less for appendages and PAX6/eyeless for
eye development; Panganiban et al. 1996; Gehring &
Ikeo 1999) most likely represent homologous genes
patterning non-homologous structures. Finally,
because most of these genes are already expressed in
cnidarians, greater care needs to be exercised whenPhil. Trans. R. Soc. Btime spans, incongruent or unresolved phylogenies
stem from systematic errors. These errors are due
to inaccuracies of the methods used in treecan be mapped onto the tree to decorate specific nodes
and branches.
4. MOLECULAR PHYLOGENY OF THE BILATERIA:
NEW DATA
In the last 10 years, molecular data have greatly changed
perspectives on the relationships of the Bilateria. The
so-called ‘new animal phylogeny’ (Adoutte et al. 2000),
initially based on 18S rDNA sequence data alone, split
the Bilateria into three superphyla: Deuterostomia,
Ecdysozoa and Lophotrochozoa, widely accepted
today. A major result was to shift all acoelomate and
pseudocoelomate groups, traditionally considered at
the base of the Bilateria, into the Ecdysozoa and
Lophotrochozoa (Adoutte et al. 2000). Further nuclear
and mitochondrial markers and combined morpho-
logical–molecular studies also support these findings
(Peterson & Eernisse 2001; Halanych 2004).
In all schemes concerning the early history of
bilaterians, the flatworms (phylum Platyhelminthes)
had a central role—their simple morphology (acoelo-
mate, non-segmented with a blind gut) coupled with a
gradualistic view of evolution made them the perfect
transitional taxon from cnidarian diploblasts to bilaterian
triploblasts. Their monophyly, however, has always been
in dispute (Smith et al. 1986). The first comprehensive
molecular trees of the Platyhelminthes and other
bilaterian and non-bilaterian phyla using 18S rDNA
sequences (Carranza et al. 1997; Ruiz-Trillo et al. 1999;
Jondelius et al. 2002) ran contrary to morphological
analysis: Platyhelminthes was polyphyletic with two of its
orders, the Acoela and the Nemertodermatida, branch-
ing as early bilaterian clades while the rest of the
Platyhelminthes (CatenulidaCRhabditophora) fell
within the Lophotrochozoa (reviewed in Bagun˜a` &
Riutort 2004a,b). Importantly, the early branching
position of acoels and nemertodermatids also contra-
dicted one of the tenets of the new animal phylogeny: the
non-basal position of acoelomate organisms. Sequences
of other nuclear genes (including HOX cluster genes)
and mitochondrial genes (Ruiz-Trillo et al. 2002, 2004;
Cook et al. 2004) corroborated this early branching
position. It is important to point out that based on
perceived morphological synapomorphies, Acoela and
Nemertodermatida are classified as sister groups forming
the taxon Acoelomorpha (Ehlers 1985). However,
because in most molecular trees they branch paraphyle-
tically (Jondelius et al. 2002; Ruiz-Trillo et al. 2004;
Wallberg et al. 2007), the monophyletic status of
the Acoelomorpha is here left open and from now on
dubbed ‘Acoelomorpha’.
Notwithstanding these advances, the cladogenetic
events at the base of the Bilateria and among most phyla
belonging to the three big superphyla remain poorly
resolved. This lack of resolution, also reproduced using
large datasets of genes and taxa, was thought to result
from the high levels of stochastic changes along
presumably closely spaced cladogenetic events such as
the origin and radiation of bilaterians (Rokas & Carroll

strot
Gastrotricha 2
Micrognathozoa
Rotifera (5
98
ta
(4)
unc
a (4
mat
ropo
ncha
mat
ata (
rata
Ne
rom
her
if m
lon
sca
4 J. Bagun˜a` et al. Phylogeny of the Bilateriareconstruction directly related to model misspecifica-
82
61
61
60
97
97
82
63
63
65
54
68
98
96
Entoproc
Nemertea
Echiura (3)
Sip
Annelid
Mollusca (5)
Brachiopoda (3)
Phoronida (2)
Ne
Arth
Kinorhy
Priapulida (2)
Urochordata
Cephalochordata
Echinoder
Hemichord
Verteb
Cnidaria (7)
0.05
Figure 2. Bayesian analysis of 18SC28S sequences (3696 nts) f
Posterior probabilities are indicated when less than 100%, ot
numbers in parentheses indicate the number of taxa sampled
support (except for gastrotrichs). Boxed groups correspond to
were carried out (see electronic supplementary material). TheGa
91
7362
83tions (Felsenstein 2004). To avoid them and to improve
tree reconstruction, several approaches are currently
available (Philippe & Telford 2006): allowing for the
numerous observed heterogeneities of the evolutionary
process in models of sequence evolution (e.g. CAT
model of Lartillot & Philippe (2004); Lartillot et al.
2007); increasing the number of taxa; removing the
fastest evolving positions from the dataset; and exclud-
ing fast-evolving taxa to avoid long-branch attraction
effects (LBA; Felsenstein 2004). Furthermore, the
so-called rare genomic changes (RGCs; Rokas &
Holland 2000; Rokas & Carroll 2006) are considered
more reliable characters than conventional linear,
homoplasy-sensitive sequences to avoid these problems
and resolve these cladogenetic events.
In what follows, we observe these guidelines for
ribosomal and nuclear gene sequences and introduce
unconventional RGCs, such as microRNAs, to bilaterian
phylogeny. Our main aims were to test again the position
of the ‘acoelomorph’ flatworms as early branching
bilaterians (Ruiz-Trillo et al. 1999, 2002) and to single
out early branching phyla at the base of the three
superphyla. Finally, and based on the growing consensus
of cnidarians as true bilaterians (Finnerty et al. 2004;
Martindale 2005), a new systematic proposal for the
Bilateria is suggested.
(a) Linear (quantitative) markers
(i) Ribosomal genes
To minimize mutational saturation and homoplasies
from ribosomal gene sequences and to avoid LBA
Phil. Trans. R. Soc. Beffects, we used methods less sensitive to LBA
)
Catenulida (2)
Acantocephala (3)
Lophotrochozoa
Ecdysozoa
Chaetognatha (2)
Deuterostomia
Acoela (3)
Rhabditophora (8)
Bryozoa
Cycliophora
la (2)
)
omorpha (3)
Nematoda (3)
Tardigrada
da (11)
a (6)
4)
(7)
mertodermatida
106 metazoan representatives with Cnidaria as the outgroup.
wise a bullet is present on the node. Phyla are collapsed and
ore than one. The monophyly of each phylum has maximum
g-branched or problematic groups for which specific analyses
le bar indicates the number of changes per site.Gnathostomulida (3)
Gastrotricha 3
richa 1(maximum likelihood and Bayesian inference), model
modifications such as rate heterogeneity across sites
and the slowest evolving taxa available. From 564
18S and 142 28S rDNA sequences, a combined
18C28S rDNA dataset of over 3700 bps was obtained
with 104 taxa for 28 bilaterian phyla and the outgroup.
A basic dataset was obtained avoiding six long-branch
(LB) phyla (Acoela, Gnathostomulida, Gastrotricha,
Acanthocephala, Bryozoa and Chaetognatha) and a
tree was built which reproduced the backbone of the
new animal phylogeny with very high support (for
further details, see electronic supplementary material).
When LB phyla were introduced into this basic tree,
either individually or in combination, the topology
remained unchanged and statistically highly supported
(figure 2; Paps et al., unpublished data). Contrary to
expectations, LB phyla did not cluster together at the
base but fell at specific places within each superphylum:
acanthocephalans with rotifers; gnathostomulids and
gastrotrichs as early branching lophotrochozoans;
bryozoans as sister group to a clade of cycliophorans
and entoprocts; chaetognaths, albeit with very low
support (bootstrap BPZ0.54), as sister group to
Scalidophora; and, most importantly, a paraphyletic
‘Acoelomorpha’ comprising Acoela and Nemertoder-
matida that were, with maximum support, the earliest
branching bilaterian clades.
(ii) Other nuclear genes
A second tree was obtained from a dataset of 13 genes
(18C28S genes and 11 nuclear genes) from 71 species

Nemertea (3)
Phylogeny of the Bilateria J. Bagun˜a` et al. 598
55
96
88
89
Echiura
Sipunculida
Lophotrochozoa
Ecdysozoa
Deuterostomia
Annelida (4)
Mollusca (7)
Phoronida
Brachiopoda (2)
Priapulida
Kinorhyncha
Nematoda (3)
Arthropoda (13)
Hemichordata (2)
Echinodermata (7)
Chordata (7)
Nemertodermatida
Acoela (4)
Cnidaria (5)Bryozoa (2)83
Rotifera (3)
Catenulida
Rhabditophora (5)of 21 bilaterian phyla and the outgroup. Figure 3
summarizes the tree drawn from the concatenated
dataset (J. Paps, J. Bagun˜a` & M. Riutort 2007,
unpublished data; see electronic supplementary
material). Of the six long-branch phyla included in the
18C28S tree, only two (Acoela and Bryozoa) could be
analysed and included here. The three superphyla
appeared again with maximal support, except for a 0.89
BP value for deuterostomes. Within each superphylum,
phyla grouped similarly, albeit more robustly, than in the
ribosomal tree. Two differences are noteworthy: first the
clustering, with low support, of Rotifera with Bryozoa
(BPZ0.83) and second, the low support (BPZ0.55) of
annelids and relatives with molluscs, phoronids and
brachiopods forming, together with nemerteans, a highly
supported Eutrochozoa group (BPZ1.00) sister to the
rhabditophoran platyhelminths. Finally, and most
importantly, ‘Acoelomorpha’ branched paraphyletically
and with maximal support at the base of the Bilateria.
(iii) Phylogenomics using expressed sequence tags
Because trees reconstructed from sequences of few
genes, even from many taxa, are prone to stochastic
errors while those with few taxa and many genes may
generate systematic errors, gathering many sequences
0.05
Figure 3. Bayesian analysis of concatenated sequences for
18S, 28S and 11 nuclear protein genes (9290 nts) from 74
metazoan representatives with Cnidaria as the outgroup.
Posterior probabilities are indicated when less than 100%,
otherwise a bullet is present on the node. Phyla are collapsed
and numbers in parentheses indicate the number of taxa
sampled if more than one. The monophyly of each phylum
has maximum support. The scale bar indicates the number of
changes per site. For further details, see electronic supple-
mentary material.
Phil. Trans. R. Soc. Bfrom many taxa might counter both sources of
errors and produce a well-resolved animal phylogeny
(Philippe & Telford 2006). From several species of
each phylum, 5000 expressed sequence tags (ESTs;
drawn from cDNA libraries) is considered a reasonable
number from which to select a set of suitable genes
that, under appropriate evolutionary models, will result
in better and more robust phylogenetic trees than those
drawn from gene- or species-poor trees.
We produced an EST collection from the acoel
Convoluta pulchra from which 68 different protein-
coding genes were unequivocally assigned to a dataset
of conserved single-copy genes from 51 species
belonging to 10 different bilaterian phyla and 4
outgroup phyla. An alignment of 11 959 amino acids
was established and trees inferred by maximum
likelihood under the standard WAG model (Whelan &
Goldman 2001) and by PHYLOBAYES analyses with the
CAT mixture model that overcomes LBA artefacts
when other models fail (Lartillot et al. 2007). Whereas
the standard WAG model groups together the two
long branches of Platyhelminthes and the acoel, the
resulting tree under the CAT model, with and without
the outgroup, strongly rejects this grouping. Platyhel-
minthes (with the exception of the acoel) fell within the
lophotrochozoans. The acoel studied instead clustered
either with all deuterostomes, to Xenoturbellida, or to
Ambulacraria. Importantly, when the outgroup was
removed, the acoel remained in a basal position, sister
group to the Xenoturbellida (Philippe et al. 2007).
This is the first time using a large set of data that
acoels were shown not to belong to the classical
Platyhelminthes, making the latter polyphyletic. The
deuterostome affinities of the acoels, however puzzling,
seem to contradict the early emergence of acoels at the
base of the Bilateria (Ruiz-Trillo et al. 1999). None-
theless, the very fast evolutionary rates shown by the
acoel C. pulchra make it not the best acoel species to
study even using the CAT method. This calls for new
data from slowly evolving acoels (and from its putative
sister group, the Nemertodermatida) to solve this
challenging phylogenetic problem.
(b) Qualitative markers
(i) HOX cluster genes
HOX and ParaHOX genes from five species of acoels
and a single nemertodermatid have recently been
isolated and analysed (Cook et al. 2004; Jime´nez-Guri
et al. 2006; Bagun˜a` et al. 2008; M. Q. Martindale
2004, personal communication). Overall, the maximal
set deduced for both taxa would consist of an anterior,
one/two central and one posterior HOX genes, and
one representative each of the Xlox and Cdx ParaHOX
genes. This putative simple HOX gene cluster in
‘acoelomorphs’ has been considered (Bagun˜a` &
Riutort 2004a) intermediate between the expanded
set (at least seven out of eight paralogy groups) of
most bilaterians and the simpler set of HOX/
ParaHOX genes in cnidarians (only anterior and
posterior HOX and ParaHOX genes reported with
no representatives of central genes; Chourrout et al.
2006; Ryan et al. 2007). Preliminary analyses of
anterior, central and posterior HOX genes in the
acoel Symsagitiffera roscoffensis using BAC libraries and

6 J. Bagun˜a` et al. Phylogeny of the Bilateriafluorescent in situ hybridization (FISH) show them
located on different chromosomes (E. Moreno,
J. Bagun˜a` & P. Martı´nez 2007, unpublished data);
therefore, the putative HOX cluster is dispersed.
Preliminary results on HOX gene expression in the
acoel Convolutriloba longifissura show a coarse collinear
expression along the A–P axis (Hejnol & Martindale
in press; M. Q. Martindale 2004, personal communi-
cation). However, until whole genome sequences of
acoels or nemertodermatids are made available, the
presence of new, undetected, HOX and ParaHOX
genes in these taxa could not be ruled out and,
therefore, the precise number and type of HOX cluster
genes will remain unsettled.
(ii) microRNA sets
The recently discovered microRNAs (miRNAs) rep-
resent new and powerful molecular markers to examine
unique genetic and/or biochemical apomorphies rela-
tively immune from homoplasy. The main phylogenetic
asset is the rough correlation between the number
of different miRNAs with both the hierarchy of metazoan
relationships and the number of differentiated cell types
as a measure of morphological complexity (Sempere
et al. 2006). When a large set of non-paralogous miRNAs
were traced along a wide range of taxa using northern
blots, 21 miRNAs were found common to protostomes
and deuterostomes of which none is present in sponges
and two in cnidarians (Sempere et al. 2006). Proto-
stomes had 12 additional specific miRNAs and deuter-
ostomes had 7. Platyhelminthes, represented by a
marine polyclad, had almost all protostome miRNAs
excluding the two ecdysozoan-specific miRNAs so far
detected, confirming that they are lophotrochozoan
protostomes. Interestingly, the sole acoel included,
Childia sp., had only a subset (six miRNAs) of the
miRNAs shared by protostomes and deuterostomes.
Recently, we examined the miRNA complement of a
second acoel taxon, Symsagitiffera roscoffensis, and three
additional rhabditophoran platyhelminth taxa, one
polyclad and two triclads. Symsagitiffera roscoffensis
possesses an identical subset of miRNAs to Childia
sp. found across protostomes and deuterostomes, and
none of the miRNAs unique to protostomes or
planarians (Sempere et al. 2007). This supports again
the polyphyly of the Platyhelminthes and that the
Acoela are early branching bilaterians. Were acoels
members of the Platyhelminthes and simply had a
reduced number of miRNAs due to secondary loss,
then one would expect acoels to bear a mosaic or ‘salt-
and-pepper’ pattern of miRNAs such that some
primitive (triploblast specific) and some, but not all,
derived (nephrozoan- and protostome-specific) miR-
NAs would be detected (Sempere et al. 2007).
(c) Summing up
This report and previous studies (reviewed in Bagun˜a` &
Riutort 2004a) are consistent with the view that acoels
and nemertodermatids are early branching bilaterian
lineages (figures 2 and 3). However, two features of
these figures need clarification. First, the conflict
between the topology of these trees (acoels and
nemertodermatids as early branching bilaterians)
and trees recovered from EST analyses (acoels asPhil. Trans. R. Soc. Bdeuterostomes; Philippe et al. 2007). Second, the
taxonomic status of acoels and nemertodermatids,
either branching paraphyletically at the base (Jondelius
et al. 2002; Ruiz-Trillo et al. 2004) dismissing the
‘Acoelomorpha’ as a valid taxon (Wallberg et al. 2007)
or, as morphologists claim (Smith et al. 1986), sister
groups forming a monophyletic Acoelomorpha.
Our EST analysis incorporated a large number of
characters, though few phyla were included and acoels
were represented by a single species that unfortunately
had very fast clock behaviour. In contrast, ribosomal
and nuclear gene analyses included fewer characters,
but they had better phyla and within-phyla sampling,
and acoelomorphs were represented by four out of five
species (which included a nemertodermatid), some
short branched. Such differences translate into striking
differences in bootstrap support values as regards
acoelomorph position: maximal for nuclear genes and
very weak in the EST analysis (see fig. 1 in Philippe
et al. 2007). Waiting for new data from slowly evolving
acoels and nemertodermatids, current evidence and
HOX cluster gene and miRNA datasets favour
the topology represented in figure 4 as regards the
phylogenetic position of the acoelomorphs and of the
acoels in particular.
Support for a monophyletic ‘Acoelomorpha’ in
molecular trees relies solely on myosin heavy chain II
sequences (Ruiz-Trillo et al. 2002). All remaining trees
and those reported here (figures 2 and 3) recover the
Acoela and the Nemertodermatida as the first two
separate branches within the Bilateria with high
support. This dismisses the ‘Acoelomorpha’ as a valid
taxon (Wallberg et al. 2007). How does this molecular
evidence fit with claimed morphological synapomor-
phies linking acoels with nemertodermatids (i.e. the
special structure of the basal body-rootlet system
complex and the ciliary tips and the fine structure of
the frontal organ; Smith et al. 1986)? Most of these
structures occur in other metazoan groups including
the Xenoturbellida, recently proposed to be a deuter-
ostome (Bourlat et al. 2006). Moreover, these
structures are only superficially similar and probably
originated by convergence. Together with differences in
sperm morphology, neurotransmitter patterns
and embryonic cleavage patterns between acoels and
nemertodermatids, most evidence is consistent with
Acoela and Nemertodermatida as separate early
branching clades and with the ‘Acoelomorpha’ as a
non-monophyletic clade (Wallberg et al. 2007).
A second important outcome from figure 2 is the
presence, albeit with moderate to low support, of
acoelomate (Gnathostomulida and Gastrotricha) and
pseudocoelomate (Rotifera) groups as early branching
lophotrochozoans. In addition, the new Platyhel-
minthes (Platyhelminthes sensu lato excluding the
acoelomorphs) either alone or with other phyla appears
as sister group to the spiralian Eutrochozoa (annelids,
molluscs and relatives) and not buried within the
Lophotrochozoa. Together with the recent placement
of the acoelomate Xenoturbellida as sister group to all
deuterostomes (Perseke et al. 2007) or to the
Ambulacraria (Bourlat et al. 2006), these data suggest
the need to re-evaluate in depth the so-called new
animal phylogeny (Adoutte et al. 2000).

LC
y
3C
Phylogeny of the Bilateria J. Bagun˜a` et al. 7LCBA
1 2
5 6
7 8 9
P/D
x
3A 4A
3B 4B5. THE BILATERIA: A NEW SYSTEMATIC
PROPOSAL
In all zoological textbooks, cnidarians (anthozoansC
medusozoans) are classified as organisms with radial
symmetry. Although in most anthozoan cnidarians
bilaterally symmetric features (e.g. slit-shaped mouth,
internal mesenteries and asymmetric siphonoglyphs in
the polyp form) were noted in the past (Stephenson
1926; Hyman 1951; Salvini-Plawen 1978; Willmer
1990), they were not taken as evidence for bilaterality
because anthozoans were considered derived cnidar-
ians and hence their internal bilateral features as
secondarily evolved. Recent molecular phylogenies,
however, have shown that Cnidaria and Bilateria are
sister groups (Medina et al. 2001; Wallberg et al. 2004)
and, importantly, that the Anthozoa is not a derived
cnidarian clade but a basal group rendering its
bilaterally symmetric features as possible plesiomor-
phies for the cnidarians (Collins 2002). Hence,
cnidarians could originally be truly bilaterian
(Finnerty et al. 2004; Martindale 2005) albeit
Figure 4. A new systematic proposal for the Bilateria. Morpho
microRNA sets) have been mapped onto a backbone tree drawn fr
includes the Cnidaria and the former Bilateria, now dubbed as Tri
(Acoela and Nemertodermatida) and the rest of the bilateria
Triploblastica is less complex than the ancestor for Triploblastica.
hatched inverted triangles) are as follows: 1, D–V axis; 2, bila
ParaHOX); 4, microRNA sets (4A: basic bilaterian set). The Trip
3B (4 Hox/3 ParaHox); 4B, a miRNA set of five out of six gene
Finally, the Nephrozoa (ZProtostomesCDeuterostomes at the P
acoelomorphs: 3C, an expanded HOX cluster gene of seven to eig
7, small anterior brain ganglia and ventral nerve cords; 8, through
As suggested by some authors, other autapomorphies of Nephro
though they may have a monophyletic or a polyphyletic origin. So
and Ecdysozoa are indicated: 4D–4G, specific miRNA sets; 12,
postulated synapomorphies for a monophyletic Acoelomorpha (S
system complex and the ciliary tips; y, fine structure of the fron
innovations appeared. See text for further details and main refere
Phil. Trans. R. Soc. B1011
12 13
10 11
10 11
10 11
Nephrozoa
Triploblastica
Bilateria
A
4C
4D
4E
4F
Lophotrochozoa
Metazoa
Ecdysozoa
Deuterostomia
Nemertodermatida
Acoela
Cnidaria
Ctenophore
Porifera
4G14secondarily modified to radiality (externally) owing to
their predominantly sessile life style.
The homology between the O–AB axis of cnidarians
and the A–P axis of bilaterians is now widely accepted,
though the precise equivalences between oral (O) and
aboral (AB) ends of cnidarians to anterior (A) and
posterior (P) ends of bilaterians are disputed (OZP
and ABZA: Salvini-Plawen 1978; Meinhardt 2002;
Rentzsch et al. 2007; Bagun˜a` et al. 2008; OZA and
ABZP: Finnerty et al. 2004; Martindale 2005; Matus
et al. 2006b). Moreover, the presumed homology of the
‘directive axis’ of cnidarians to the bilaterian D–V axis,
initially based on the transient asymmetric expression
of the cnidarian orthologues of BMP2/4/dpp and other
D–V genes, has been amply corroborated by the asym-
metric expressions of scores of ‘endodermal’, ‘meso-
dermal’ and ‘neural’ genes (reviewed in Martindale
2005; Matus et al. 2006b).
The increasing evidence of cnidarians as bilateral
in origin, new molecular data (figures 2 and 3 and
microRNA datasets) backing the acoelomorph
Placozoa
Choanoflagellida
logical and molecular characters (HOX cluster genes and
om 18SC28S rDNA and 11 nuclear genes. The new Bilateria
ploblastica, the latter split into a paraphyletic ‘Acoelomorpha’
ns or Nephrozoa. Note that the LCBA for cnidariansC
Bilaterian autapomorphies (vertical solid lines and empty and
teral symmetry; 3, HOX/ParaHOX clusters (3A: 2 HOX/2
loblastica have some autapomorphies that exclude cnidarians:
s; 5, mesoderm; 6, clustered nerve cells at the anterior end.
/D LCA node) will have some autapomorphies that exclude
ht genes; 4C, a nephrozoan miRNA set of 20 or more genes;
gut (mouthCanus); 9, excretory system (Zprotonephridia).
zoa would be: 10, coelomic cavities; 11, body segmentation,
me autapomorphies for the Deuterostomia, Lophotrochozoa
post-anal tail; 13, gill slits; 14, ecdysis. x, y (broken lines),
mith et al. 1986). x, special structure of the basal body-rootlet
tal organ. Grey ovals indicate the stem branches where key
nces.

8 J. Bagun˜a` et al. Phylogeny of the Bilateriaflatworms as the earliest extant branching bilaterian
and the presence of acoelomate/pseudocoelomate
groups at the base of the lophotrochozoans and
deuterostomes prompt us to suggest a new systematic
proposal for the origin and evolution of the Bilateria
(figure 4). Under this proposal, Cnidaria are
considered true bilaterians and the sister group to a
less-inclusive bilaterian clade, here named Triplo-
blastica, which comprises present-day bilaterians with
a true mesoderm. Within the Triploblastica, molecu-
lar evidence (figures 2 and 3) favours the early
branching of a paraphyletic ‘Acoelomorpha’ (acoels
first and nemertodermatids second) sister group to
the traditional protostomeCdeuterostome clade, or
Nephrozoa (sensu Jondelius et al. 2002). Apomorphies
of the new Bilateria would be the establishment and
consolidation of a new D–V axis and the ensuing
bilateral symmetry, and the appearance of a basic
HOX cluster (2 HOX/2 ParaHOX genes) and a
minimal set (two out of three genes) of miRNAs.
Plesiomorphies of the new Bilateria, shared with the
ctenophores, would be an A–P axis (O–AB in
cnidarians and ctenophores), diploblasty (ecto-
dermCendoderm), the presence of muscle cells not
forming a true mesoderm (Burton 2008) and the
presence of a nerve net. Another key apomorphy of
Triploblastica is the clustering of nerve cells at the
anterior end from which longitudinal bundles of nerve
fibres spring. Such characters probably run parallel to
the first expansion of HOX/ParaHOX clusters (group
3 and central HOX genes; character 3B) and new
miRNAs (character 4B). After acoelomorphs and
other extant (Xenoturbellida) and extinct acoelomate/
pseudocoelomate groups split, the radiation of
Nephrozoa resulted in protonephridia, a through
gut, and the progressive development of a more
concentrated nervous system (layers of nerve cells
surrounding a neuropile and defined as ventral nerve
chords). Because the last scenario led to the
appearance of true organs and more elaborate A–P
and D–V axial patterns, full sets of HOX cluster genes
(character 3C) and new sets of miRNAs (characters
4C–4G) were also required.
The name Planulozoa was recently proposed by
Wallberg et al. (2004) to define a clade comprising
Cnidaria and Bilateria. As such, Planulozoa is formally
equivalent to the name Bilateria, here proposed for the
more inclusive clade (CnidariaCTriploblastica).
Suggested synapomorphies for the Planulozoa are the
presence of endodermal myoepithelial musculature,
septate junctions in epithelial cells, symmetrically
arranged spermatozoon heads with a mid-piece and a
set of several clustered HOX genes (Wallberg et al.
2004). Such features, however, are plesiomorphic or
have not been tested in other phyla, and, when referring
to HOX clustering in cnidarians, seem at odds with
most recent genomic data (Chourrout et al. 2006;
Ryan et al. 2007). Moreover, the name Planulozoa
stems from the presumed similarities between the
planula larva and acoel worms, both being vermiform
and having an apparent polar development of the
nervous system (for specific references, see Wallberg
et al. 2004). Again such characters are weakly defined,
have to be tested in other phyla and refer to aPhil. Trans. R. Soc. Bhypothetical process contemplated in the planuloid–
acoeloid theory. Instead, the new Bilateria here
proposed is defined by specific descriptive characters
(D–V axis, bilateral symmetry and microRNA sets).
The first asset of the proposal set forth over that
contemplated in the new animal phylogeny (see
figure 1 for comparison) is that key changes or
innovations in bilaterian evolution are spread along
several stem branches allowing character states to be
polarized. In particular, it unlinks D–V axis forma-
tion from mesoderm formation: the first appearing in
the last common ancestor (equal to LCBA) of
Cnidarians and Triploblastica and the second origi-
nated in the LCA of Triploblastica. It has been
claimed that some members of the cnidarian
Medusozoa possess a mesodermal derivative, the
entocodon (Seipel & Schmid 2005), and that
members of both Cnidaria and Ctenophora possess
striated muscle, a mesodermal derivative (Seipel &
Schmid 2006). This would imply that the last
common ancestor of Ctenophores and CnidariaC
Bilateria had already been a triploblast bearing
striated muscle (Martindale et al. 2004; Seipel &
Schmid 2005; Boero et al. 2007). However, striated
muscle in cnidarians, namely in anthozoans, is
epitheliomuscular; the entocodon and the mesoderm
have very different developmental origins (the first
from ectoderm and the second from the endoderm);
and striated muscles in ctenophores, while truly
muscular, non-epithelial and derived from the
endoderm, are very distinct from triploblastic
striated muscles (reviewed in Burton 2008).
Therefore, the more parsimonious scenario is
that the LCBA in figure 4 was a diploblast and that
triploblastic mesoderm, cnidarian entocodon and
striated musculature in Cnidaria, Ctenophora
and Triploblastica had independent origins. Under
this scenario, it could be predicted that genes
involved in mesodermal patterning and differen-
tiation in triploblasts (i.e. snail, twist, forkhead,
brachyury, mef2 and GATA) are primarily linked
with the endoderm in diploblasts, and that patterning
genes involved in muscle development within each
lineage and in the formation of the hydrozoan
entocodon (i.e. mef2, Id, msx) bear little or no
similarity in expression. Both predictions are borne
out from recent molecular data (Finnerty et al. 2004;
Martindale et al. 2004; Burton 2008). This strength-
ens the view of separate, independent origins for
muscle cells in the three clades, and for the origin of
mesoderm in triploblasts from the bipotential endo-
derm (equal to mesoendoderm) of the LCBA.
Therefore, the presence of a D–V axis in the LCBA
unlinks character 2 (D–V axis) from character 5
(mesoderm), pointing to cnidarians as the group of
choice to analyse the origins of the D–V axis and
bilateral symmetry, and to acoels and nemertoder-
matids to explore the origins of mesoderm and of a
more centralized nervous system.
The second asset of the phylogenetic tree in figure 4
is the closer similarities between the new LCBA and
the ancestor envisaged in the planuloid–acoeloid
theory than between the former and the complex
Urbilateria postulated in the archicoelomate theory.

new positions that will force a re-evaluation of the
new animal phylogeny (Adoutte et al. 2000). Finally,
De Robertis, E. M. & Sasai, Y. 1996 A common plan for
dorsoventral patterning in Bilateria. Nature 380, 37–40.
Phylogeny of the Bilateria J. Bagun˜a` et al. 9and most importantly, the growing consensus to
consider the Cnidaria bilaterally symmetric in origin
(Finnerty et al. 2004; Martindale 2005) leads us to
suggest a new systematics for the Bilateria, which
considers the Cnidaria as bilaterians and sister group to
the rest of Bilateria, now dubbed as Triploblastica to
indicate the appearance of mesoderm as one of the
most important events in animal evolution.
In the upcoming years, refinements in data acqui-
sition, evolutionary models, fossil record, molecular
phylogenies, gene expression data (see expression of
developmental genes in embryos of acoels, Hejnol &
Martindale in press) and functional evo–devo studies
will be instrumental to test the soundness of the new
proposal as well as to sort out the sequential evolution
of clades at the base of the Deuterostomia, the
Ecdysozoa and the Lophotrochozoa.
We thank the Royal Society and the organizers Tim Little-
wood and Max Telford. Some of the data included stem from
collaborative work with Herve´ Philippe’s and Kevin J.
Peterson’s laboratories. We are especially grateful to Mark
Martindale for being an endless source of ideas and
information and for sharing unpublished data, and toUnless acoels and nemertodermatids are shown to be
ancestral but simplified or just derived bilaterians, the
new LCBA leads to a smooth morphological and
developmental transition from a bilateral, diploblastic
planuloid to a bilateral, triploblast acoeloid, and from
the latter to more complex higher bilaterians. Alter-
natively, bilateral symmetry may have evolved under
selective pressure for improved internal circulation in a
cnidarian–bilaterian ancestor, inferred to be a sessile,
bilaterally symmetrical animal (Finnerty 2005).
Additional internal manifestations of bilateral sym-
metry evolved subsequently in bilaterians. As for most
proposals on the origin of bilateral organisms
with directive locomotion based on the enterocoel–
archicoelomate hypotheses, the main stumbling blocks
for its acceptance are the undefined developmental
mechanisms and the uncertain functional continuity
of intermediates between a sessile ancestor and a
benthic crawling descendent. This makes it more
plausible that, as stated in the planuloid–acoeloid
theory, bilaterality first originated in small, bottom-
dwelling organisms.
6. SUMMARY AND PROSPECTS
Phylogenetic analysis using molecular markers, under
strict conditions to avoid stochastic and systematic
errors, have corroborated the position of acoel and
nemertodermatid flatworms as the earliest extant
branching members of the Bilateria. This reinforces
the planuloid–acoeloid theories that see stem bilater-
ians as stocks of small, benthic, simple organisms
probably derived from planuloid-like organisms and
from which present-day cnidarians also probably arose.
In addition, new molecular data are helping to over-
come the simple subdivision of Bilateria into the three
large and poorly internally resolved superclades
introducing ‘minor’ phyla (e.g. Gnathostomulida,
Gastrotricha, Chaetognatha, Xenoturbellida) intoPhil. Trans. R. Soc. B(doi:10.1038/380037a0)
Donoghue, M. J. 2005 Key innovations, convergence
and success: macroevolutionary lessons from plant
phylogeny. Paleobiology 31, 77–93. (doi:10.1666/0094-
8373(2005)031[0077:KICASM]2.0.CO;2)
Easteal, S. 1985 Generation time and the rate of molecular
evolution. Mol. Biol. Evol. 2, 450–453.
Ehlers, U. 1985 Das Phylogenetische System der Plathelminthes.
Stuttgart, Germany; New York, NY: Gustav Fischer.Reinhard Rieger, Hans Meinhardt, Jordi Garcı´a-Ferna`ndez,
Bert Hobmayer, Hiroshi Shimizu and Uli Technau for their
lively discussions and stimulating insights on the subject.
Constructive criticisms from reviewers, which greatly helped
to improve the manuscript, are warmly acknowledged. These
studies were supported by grants from the Generalitat de
Catalunya and from CICYT (Ministerio de Ciencia y
Tecnologia) to J.B., M.R. and P.M.
REFERENCES
Abouheif, E. 1997 Developmental genetics and homology: a
hierarchical approach. Trends Ecol. Evol. 12, 405–408.
(doi:10.1016/S0169-5347(97)01125-7)
Adoutte, A., Balavoine, G., Lartillot, N., Lespinet, O.,
Prud’homme, B. & de Rosa, R. 2000 The new animal
phylogeny: reliability and implications. Proc. Natl Acad.
Sci. USA 97, 4453–4456. (doi:10.1073/pnas.97.9.4453)
Arendt, D. & Nu¨bler-Jung, K. 1994 Inversion of dorsoventral
axis? Nature 371, 26. (doi:10.1038/371026a0)
Bagun˜a`, J. & Riutort, M. 2004a The dawn of bilaterian
animals: the case of the acoelomorph flatworms. BioEssays
26, 1046–1057. (doi:10.1002/bies.20113)
Bagun˜a`, J. & Riutort, M. 2004b Molecular phylogeny of the
Platyhelminthes. Can. J. Zool. 82, 168–193. (doi:10.1139/
z03-214)
Bagun˜a`, J., Martinez, P., Paps, J. & Riutort, M. 2008
Unravelling body-plan and axial evolution in the Bilateria
with molecular phylogenetic markers. In Evolving
pathways: key themes in evolutionary developmental biology
(eds A. Minelli & G. Fusco), pp 213–235. Cambridge,
UK: Cambridge University Press.
Boero, F., Schierwater, B. & Piraino, S. 2007 Cnidarian
milestones in metazoan evolution. Integr. Comp. Biol. 47,
693–700. (doi:10.1093/icb/icm041)
Bourlat, S. J. et al. 2006 Deuterostome phylogeny reveals
monophyletic chordates and the new phylum Xenoturbel-
lida. Nature 444, 85–88. (doi:10.1038/nature05241)
Burton, P. M. 2008 Insights from diploblasts; the evolution
of mesoderm and muscle. J. Exp. Zool. Mol. Dev. Evol. B.
310B, 5–14. (doi:10.1002/jez.b.21150)
Carranza, S., Bagun˜a`, J. & Riutort, M. 1997 Are the
Platyhelminthes a monophyletic primitive group? An
assessment using 18S rDNA sequences. Mol. Biol. Evol.
14, 485–497.
Carroll, S. B., Grenier, J. K. & Weatherbee, S. D. 2001 From
DNA to diversity. Malden, MA: Blackwell Science.
Cavalier-Smith, T. 1985 The evolution of genome size. New
York, NY: Wiley.
Chourrout, D. et al. 2006 Minimal ProtoHox cluster inferred
from bilaterian and cnidarian Hox complements. Nature
442, 684–687. (doi:10.1038/nature04863)
Collins, A. G. 2002 Phylogeny of Medusozoa and the
evolution of cnidarian life cycles. J. Evol. Biol. 15,
418–432. (doi:10.1046/j.1420-9101.2002.00403.x)
Cook, C. E., Jime´nez, E., Akam, M. & Salo´, E. 2004 The Hox
gene complement of acoel flatworms, a basal bilaterian
clade. Evol. Dev. 6, 154–163. (doi:10.1111/j.1525-142X.
2004.04020.x)

10 J. Bagun˜a` et al. Phylogeny of the BilateriaFelsenstein, J. 2004 Inferring phylogenies. Sunderland, MA:
Sinauer Associates, Inc.
Finnerty, J. R. 2005 Did internal transport, rather than direct
locomotion, favor the evolution of bilateral symmetry
in animals? BioEssays 27, 1174–1180. (doi:10.1002/
bies.20299)
Finnerty, J. R., Pang, K., Burton, P., Paulson, D. &
Martindale, M. Q. 2004 Origins of bilateral symmetry:
Hox and dpp expression in a sea anemone. Science 304,
1335–1337. (doi:10.1126/science.1091946)
Gehring, W. J. & Ikeo, K. 1999 Pax 6, mastering eye
morphogenesis and eye evolution. Trends Genet. 15,
371–376. (doi:10.1016/S0168-9525(99)01776-X)
Hahn, M. W. & Wray, G. A. 2002 The G-value paradox. Evol.
Dev. 4, 73–75. (doi:10.1046/j.1525-142X.2002.01069.x)
Halanych, K. M. 2004 The new view of animal phylogeny.
Annu. Rev. Ecol. Evol. Syst. 35, 229–256. (doi:10.1146/
annurev.ecolsys.35.112202.130124)
Hejnol, A. & Martindale, M. Q. In press. Acoel development
supports a simple planula-like Urbilaterian. Phil. Trans. R.
Soc. B 363. (doi:10.1098/rstb.2007.2239)
Holland, N. D. 2003 Early central nervous system evolution:
an era of skin brains? Nat. Rev. Neurosci. 4, 1–11. (doi:10.
1038/nrn1175)
Hyman, L. H. 1951 The invertebrates. Platyhelminthes and
Rhynchocoela, vol. II. New York, NY: McGraw-Hill.
Jime´nez-Guri, E., Paps, J., Garcı´a-Ferna`ndez, J. & Salo´, E.
2006 Hox and ParaHox genes in Nemertodermatida, a
basal bilaterian clade. Int. J. Dev. Biol. 50, 675–679.
(doi:10.1387/ijdb.062167ej)
Jondelius, U., Ruiz-Trillo, I., Bagun˜a`, J. & Riutort, M. 2002
The Nemertodermatida are basal bilaterians and not
members of the Platyhelminthes. Zool. Scr. 31, 201–215.
(doi:10.1046/j.1463-6409.2002.00090.x)
Kimmell, C. B. 1996 Was Urbilateria segmented? Trends Genet.
12, 329–331. (doi:10.1016/S0168-9525(96)80001-1)
Kimura, M. & Ohta, T. 1973 Eukaryotes–prokaryotes
divergence estimated by 5S ribosomal RNA sequences.
Nature 243, 199–200. (doi:10.1038/243199a0)
Lartillot, N. & Philippe, H. 2004 A Bayesian mixture model
for across-site heterogeneities in the amino-acid replace-
ment process. Mol. Biol. Evol. 21, 1095–1109. (doi:10.
1093/molbev/msh112)
Lartillot, N., Brinkmann, H. & Philippe, H. 2007 Suppres-
sion of long-branch attraction artefacts in the animal
phylogeny using a site-heterogeneous model. BMC Evol.
Biol. 7(Suppl. 1), S4. (doi:10.1186/1471-2148-7-S1-S4)
Martindale, M. Q. 2005 The evolution of metazoan axial
properties. Nat. Rev. Genet. 6, 917–927. (doi:10.1038/
nrn1818)
Martindale, M. Q., Pang, K. & Finnerty, J. R. 2004
Investigating the origins of triploblasty: ‘mesodermal’
gene expression in a diploblastic animal, the sea anemone
Nematostella vectensis (phylum, Cnidaria; class, Anthozoa).
Development 131, 2463–2474. (doi:10.1242/dev.01119)
Matus, D. Q., Thomsen, G. H. & Martindale, M. Q. 2006a
Dorso/ventral genes are asymmetrically expressed and
involved in germ-layer demarcation during cnidarian
gastrulation. Curr. Biol. 16, 499–505. (doi:10.1016/
j.cub.2006.01.052)
Matus, D. Q., Pang, K., Marlow, H., Dunn, C. W., Thomsen,
G. H. & Martindale, M. Q. 2006b Molecular evidence for
deep evolutionary roots of bilaterality in animal develop-
ment. Proc. Natl Acad. Sci. USA 103, 11 195–11 200.
(doi:10.1073/pnas.0601257103)
Medina, M., Collins, A. G., Silberman, J. D. & Sogin, M. L.
2001 Evaluating hypotheses of basal animal phylogeny
using complete sequences of large and small rRNA. Proc.
Natl Acad. Sci. USA 98, 9707–9712. (doi:10.1073/
pnas.171316998)Phil. Trans. R. Soc. BMeinhardt, H. 2002 The radial-symmetric hydra and the
evolution of the bilateral body plan: an old body became a
young brain. BioEssays 24, 185–191. (doi:10.1002/
bies.10045)
Nei, M. & Kumar, S. 2000 Molecular evolution and
phylogenetics. Oxford, UK: Oxford University Press.
Panganiban, G. E. F. et al. 1996 The origin and evolution of
animal appendages. Proc. Natl Acad. Sci. USA 94,
5162–5166. (doi:10.1073/pnas.94.10.5162)
Perseke, M., Hankeln, T., Weich, B., Fritzsch, G., Stadler,
P. F., Israelsson, O., Bernhard, D. & Schlegel, M. 2007
The mitochondrial DNA of Xenoturbella bocki: genomic
architecture and phylogenetic analysis. Theory Biosci. 126,
35–42. (doi:10.1007/s12064-007-0007-7)
Peterson, K. J. & Eernisse, D. J. 2001 Animal phylogeny and
the ancestry of bilaterians: inferences from morphology
and 18S rDNA gene sequences. Evol. Dev. 3, 170–205.
(doi:10.1046/j.1525-142x.2001.003003170.x)
Philippe, H. & Telford, M. J. 2006 Large-scale sequencing
and the new animal phylogeny. Trends Ecol. Evol. 21,
614–620. (doi:10.1016/j.tree.2006.08.004)
Philippe, H., Brinkmann, H., Martı´nez, P., Riutort, M. &
Bagun˜a`, J. 2007 Acoel flatworms are not Platyhelminthes:
evidence from phylogenomics. PLoS ONE 2, e717.
(doi:10.1371/journal.pone.0000717)
Raff, R. A. 2000 Evo–devo: the evolution of a new discipline.
Nat. Rev. Genet. 1, 74–79. (doi:10.1038/35049594)
Remane, A. 1963 The enterocoelic origin of the coelom. In
The lower Metazoa (ed. E. C. Dougherty), pp. 78–90.
Berkeley, CA: University of California Press.
Rentzsch, F., Anton, R., Saina, M., Hammerschmidt, M.,
Holstein, T. W. & Technau, U. 2006 Asymmetric
expression of the BMP antagonists chordin and gremlin in
the sea anemone Nematostella vectensis: implications for the
evolution of axial patterning. Dev. Biol. 296, 375–387.
(doi:10.1016/j.ydbio.2006.06.003)
Rentzsch, F., Guder, C., Vocke, D., Hobmayer, B. &
Holstein, T. W. 2007 An ancient chordin-like gene in
organizer formation of Hydra. Proc. Natl Acad. Sci. USA
104, 3249–3254. (doi:10.1073/pnas.0604501104)
Rokas, A. & Carroll, S. B. 2006 Bushes in the tree of life.
PLoS Biol. 4, e352. (doi:10.1371/journal.pbio.0040352)
Rokas, A. & Holland, P. W. H. 2000 Rare genomic changes as
a tool for phylogenetics. Trends Ecol. Evol. 15, 454–459.
(doi:10.1016/S0169-5347(00)01967-4)
Ruiz-Trillo, I., Riutort, M., Littlewood, D. T. J., Herniou, E. A.
& Bagun˜a`, J. 1999 Acoel flatworms: earliest extant Bilaterian
Metazoans, not members of Platyhelminthes. Science 283,
1919–1923. (doi:10.1126/science.283.5409.1919)
Ruiz-Trillo, I., Paps, J., Loukota, M., Ribera, C., Jondelius,
U., Bagun˜a`, J. & Riutort, M. 2002 A phylogenetic analysis
of myosin heavy chain type II sequences corroborates that
Acoela and Nemertodermatida are basal bilaterians. Proc.
Natl Acad. Sci. USA 99, 11 246–11 251. (doi:10.1073/
pnas.172390199)
Ruiz-Trillo, I., Riutort, M., Fourcade, H. M., Bagun˜a`, J. &
Boore, J. L. 2004 Mitochondrial genome data support
the basal position of Acoelomorpha and the polyphyly of
the Platyhelminthes. Mol. Phylogenet. Evol. 33, 321–332.
(doi:10.1016/j.ympev.2004.06.002)
Ryan, J. F., Mazza, M. E., Pang, K., Matus, D. Q., Baxevanis,
A. D., Martindale, M. Q. & Finnerty, J. R. 2007
Pre-Bilaterian origins of the Hox cluster and the Hox code:
evidence from the sea anemone, Nematostella vectensis. PLoS
ONE 2, e153. (doi:10.1371/journal.pone.0000153)
Salvini-Plawen, L. v. 1978 On the origin and evolution of the
lower Metazoa. Z. Zool. Syst. Evol. 16, 40–88.
Seipel, K. & Schmid, V. 2005 Evolution of striated muscle
jellyfish and the origin of triploblasty. Dev. Biol. 282,
14–26. (doi:10.1016/j.ydbio.2005.03.032)

Seipel, K. & Schmid, V. 2006 Mesodermal anatomies in
cnidarian polyps and medusae. Int. J. Dev. Biol. 50,
589–599. (doi:10.1387/ijdb.062150ks)
Sempere, L., Cole, C. N., McPeek, M. A. & Peterson, K. J.
2006 The phylogenetic distribution of Metazoan micro-
RNAs: insights into evolutionary complexity and con-
straint. J. Exp. Zool. (Mol. Dev. Evol.) B 306, 575–588.
(doi:10.1002/jez.b.21118)
Sempere, L. F., Martı´nez, P., Cole, C., Bagun˜a`, J. &
Peterson, K. J. 2007 Phylogenetic distribution of
microRNAs supports the basal position of acoel flat-
worms and the polyphyly of Platyhelminthes. Evol. Dev.
9, 409–415.
Smith III, J. P. S., Tyler, S. & Rieger, R. M. 1986 Is the
Turbellaria polyphyletic? Hydrobiologia 132, 13–21.
(doi:10.1007/BF00046223)
Stephenson, T. A. 1926 British sea anemones. London, UK:
The Ray Society.
Valentine, J. W. 2006 Ancestors and urbilateria. Evol. Dev. 8,
391–393. (doi:10.1111/j.1525-142X.2006.00112.x)
Wagner, G. P. 2007 The developmental genetics of homology.
Nat. Rev. Genet. 8, 473–479. (doi:10.1038/nrg2099)
Wallberg, A., Thollesson, M., Farris, J. S. & Jondelius, U.
2004 The phylogenetic position of the comb jellies
(Ctenophora) and the importance of taxonomic sampling.
Cladistics 20, 558–578. (doi:10.1111/j.1096-0031.2004.
00041.x)
Wallberg, A., Curini-Galletti, M., Ahmadzadeh, A. &
Jondelius, U. 2007 Dismissal of Acoelomorpha: Acoela
and Nemertodermatida are separate early bilaterian
clades. Zool. Scr. 36, 509–523. (doi:10.111/j.1463-6409.
2007.00295.x)
Whelan, A. & Goldman, N. 2001 A general empirical model
of protein evolution derived from multiple protein families
using a maximum-likelihood approach. Mol. Biol. Evol.
18, 691–699.
Willmer, P. 1990 Invertebrate relationships. Patterns in
animal evolution. Cambridge, UK: Cambridge University
Press.
Zuckerkandl, E. & Pauling, L. 1965 Evolutionary divergence
and convergence in proteins. In Evolving genes and proteins
(eds V. Bryson & H. J. Vogel), pp. 97–166. New York, NY:
Academic Press.
Phylogeny of the Bilateria J. Bagun˜a` et al. 11Phil. Trans. R. Soc. B