To Be or Not to Be a Flatworm: The Acoel Controversy
Bernhard Egger1., Dirk Steinke2., Hiroshi Tarui3., Katrien De Mulder4., Detlev Arendt5, Gae ¨tan
Borgonie4, Noriko Funayama8, Robert Gschwentner1, Volker Hartenstein6, Bert Hobmayer1, Matthew
Hooge7, Martina Hrouda8, Sachiko Ishida9, Chiyoko Kobayashi3,10, Georg Kuales1, Osamu Nishimura3,
Daniela Pfister1, Reinhard Rieger1, Willi Salvenmoser1, Julian Smith, III11, Ulrich Technau12, Seth Tyler7*,
Kiyokazu Agata8*, Walter Salzburger13*, Peter Ladurner1*
1Institute of Zoology and Center for Molecular Biosciences, University of Innsbruck, Innsbruck, Austria, 2Biodiversity Institute of Ontario, University of Guelph, Guelph,
Ontario, Canada, 3Evolutionary Regeneration Group, Center for Developmental Biology, RIKEN Kobe, Kobe, Japan, 4Department of Biology, Nematology Section,
University of Ghent, Ghent, Belgium, 5Developmental Biology Programme, EMBL, Heidelberg, Germany, 6Department of Molecular, Cell and Developmental Biology,
University of California Los Angeles, Los Angeles, California, United States of America, 7School of Biology and Ecology, University of Maine, Orono, Maine, United States of
America, 8Department of Biophysics, Graduate School of Science, Kyoto University, Kyoto, Japan, 9Department of Biofunctional Science, Faculty of Agriculture and Life
Sciences, Hirosaki University, Hirosaki, Japan, 10Division of Integrative Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto,
Japan, 11Department of Biology, Winthrop University, Rock Hill, South Carolina, United States of America, 12Department for Molecular Evolution and Development,
Centre for Organismal Systems Biology, Faculty of Life Sciences, University of Vienna, Vienna, Austria, 13Zoological Institute, University of Basel, Basel, Switzerland
Since first described, acoels were considered members of the flatworms (Platyhelminthes). However, no clear
synapomorphies among the three large flatworm taxa - the Catenulida, the Acoelomorpha and the Rhabditophora -
have been characterized to date. Molecular phylogenies, on the other hand, commonly positioned acoels separate from
other flatworms. Accordingly, our own multi-locus phylogenetic analysis using 43 genes and 23 animal species places the
acoel flatworm Isodiametra pulchra at the base of all Bilateria, distant from other flatworms. By contrast, novel data on the
distribution and proliferation of stem cells and the specific mode of epidermal replacement constitute a strong
synapomorphy for the Acoela plus the major group of flatworms, the Rhabditophora. The expression of a piwi-like gene not
only in gonadal, but also in adult somatic stem cells is another unique feature among bilaterians. These two independent
stem-cell-related characters put the Acoela into the Platyhelminthes-Lophotrochozoa clade and account for the most
parsimonious evolutionary explanation of epidermal cell renewal in the Bilateria. Most available multigene analyses produce
conflicting results regarding the position of the acoels in the tree of life. Given these phylogenomic conflicts and the
contradiction of developmental and morphological data with phylogenomic results, the monophyly of the phylum
Platyhelminthes and the position of the Acoela remain unresolved. By these data, both the inclusion of Acoela within
Platyhelminthes, and their separation from flatworms as basal bilaterians are well-supported alternatives.
Citation: Egger B, Steinke D, Tarui H, De Mulder K, Arendt D, et al. (2009) To Be or Not to Be a Flatworm: The Acoel Controversy. PLoS ONE 4(5): e5502.
Editor: Pawel Michalak, University of Texas Arlington, United States of America
Received September 25, 2008; Accepted March 24, 2009; Published May 11, 2009
Copyright: ? 2009 Egger et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: B.E. was supported by FWF P16618 and P19232, the University of Innsbruck, and the Francqui Fondation, Belgium. D.S. was funded by grants from
NSERC. K.D.M was supported by a predoctoral FWO (Belgium) fellowship. D.P. and P.L. were supported by FWF P18099 and P.L. by APART 10841. K.A. was in part
supported by the GlobalCOE Program from the Ministry of Education, Culture, Sports, Science and Technology of Japan. J.S.III was supported by the Winthrop
Research Council and the Elizabeth King Fund. S.T. was supported by NSF grant DEB-0118804. W.S. was supported by the European Research Council (ERC). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org (ST); email@example.com (KA); firstname.lastname@example.org (WS); email@example.com (PL)
. These authors contributed equally to this work.
Flatworms (phylum Platyhelminthes) have long been considered
the most basal bilaterians, and they have served as models for the
bilaterian ancestor in a variety of phylogenetic hypotheses.
Generally, morphological data place the Acoela within the
Platyhelminthes based on a combination of weak characters: an
acoelomate body structure, a densely multiciliated monolayered
epidermis leading to a common habitus, a frontal organ, neoblasts,
hermaphroditic reproduction with similar reproductive-organ
morphology, biflagellate sperms with inverted axonemes (in acoels
and rhabditophorans except macrostomorphans), and lack of
hindgut and anus [1–3]. But already one year after the most
comprehensive morphological phylogenetic system of the Platy-
helminthes was published , the monophyly of the group was
questioned on the basis of the ambiguity of the uniting characters
and because of the absence of outgroups in the assessment of the
suitability of these characters as apomorphies . In molecular
phylogenetic analyses the position of acoels remained unresolved
as well: Acoels are placed well outside the Platyhelminthes as a
sister group to the other bilaterians based on data from a single
gene or a few loci only, such as 18S and 28S rDNA, Hox and
ParaHox genes, myosin II, or microRNA [5–11]. In multigene
analyses, acoels appear within the Lophotrochozoa , or they
are associated with deuterostomes , or they are basal
bilaterians . Because morphological characters are incongru-
PLoS ONE | www.plosone.org1 May 2009 | Volume 4 | Issue 5 | e5502
ent with the various molecular phylogenetic hypotheses, the
placement of Acoela remains controversial; previous attempts to
subsume molecular and morphological data proved unsatisfactory
(reviewed in ).
We have succeeded in finding two strong synapomorphies
between acoel and rhabditophoran flatworms. The stem cell
system and the particular mode of replacing epidermal cells
represent unique features shared by both acoel and rhabdito-
phoran flatworms, but not by any other bilaterian lineage. At the
same time, our phylogenomic data support a separation of acoels
from rhabditophoran flatworms.
Results and Discussion
Phylogenomics place the Acoela at the base of the
Here, we provide new molecular and developmental data
having a bearing on the flatworm controversy. We produced ESTs
from several species: the cnidarian Aurelia aurita and Nematostella
vectensis, the sponge Ephydatia fluviatilis, the acoel Isodiametra pulchra,
the flatworm Macrostomum lignano, and the annelid Platynereis
dumerilii. Applying a phylogenomic approach on the basis of
10,218 amino acid positions of the acoel Isodiametra pulchra, we first
identified a set of open reading frames homologous to sequences
we generated from major animal taxa or which were represented
in public databases. To avoid the use of paralogs, we limited our
selection of genes to e-values #10250in blast searches. We
subjected the resulting multi-locus datasets to phylogenetic
analyses using Maximum likelihood and Bayesian inference and
analyzed two datasets consisting of 23 species represented by 43
loci (Fig. 1) or 24 species represented by 32 loci (Fig. S1, same
dataset, but including the rhabditophoran Macrostomum lignano),
representing diverse animal phyla and including partial sequence
data (see Supplementary Material and Tables S1 and S2).
In contrast to two previous multi-locus approaches [12–13], our
new molecular phylogeny puts the acoels basal to all other
bilaterians (Fig. 1, Fig. S1). The remaining flatworms consistently
appear at the base of the Lophotrochozoa and close to coelomate
spiralian phyla such as Mollusca and Annelida (cf., e.g. ). Our
multi-locus phylogenetic analysis suggests, as others have, a
separation of acoels from rhabditophoran flatworms and a sister-
group relationship of acoels to the remaining bilaterians. Apart
from the position of the acoels, the overall topology of the tree
inferred by our approach is congruent with the current view of
animal evolution [12–15].
The unique stem cell system unites acoel and
For the analysis of novel developmental data, we focused on the
extraordinary stem cell system of flatworms. We mapped the
distribution of S-phase stem cells and epidermal replacement in
acoel flatworms using three species from two families (Fig. 2), and
in rhabditophoran flatworms including four species from two
orders and four different families (Fig. 3). We also included, for
comparative reasons, the annelid Dorvillea bermudensis and the
nemertean Cephalothrix sp. in the analysis of the distribution of S-
phase stem cells (Fig. 4). Additionally, we performed in situ
hybridization experiments with the stem-cell-specific marker piwi
in the acoel flatworm I. pulchra and the rhabditophoran flatworm
M. lignano (Fig. 5).
Our experiments clearly demonstrate that, in acoels, epidermal
cells are exclusively renewed from mesodermally located stem cells
(Fig. 2). The very same mode of epidermal cell renewal and the
absence of proliferating cells in the epidermis characterizes
rhabditophoran taxa such as macrostomorphans  (Fig. 3),
polyclads  (Fig. 3), triclads , rhabdocoels  and parasitic
platyhelminths . In contrast, proliferating cells in the epidermis
occur in all other lophotrochozoans investigated, including
annelids  (Fig. 4A–C), nemertines (Fig. 4D–E), and molluscs
. Thus, it is the nature of epidermal replacement - through
stem cells originating from the mesodermal space rather than the
epidermis itself - that sets acoels and rhabditophorans apart from
other bilaterian taxa.
Gene expression patterns of the stem-cell marker piwi further
substantiate the acoel-rhabditophoran grouping. Within the
Bilateria, piwi-like genes are highly evolutionarily conserved, and
expression is largely restricted to the germline, where it plays an
important role in germ-cell development and maintenance, in
meiosis, as well as in the regulation of retrotransposons [23–24]. In
most animals studied so far, piwi RNA interference results in
sterility. However, in triclads, as well as in M. lignano and I. pulchra,
piwi-like gene expression is extended to a subpopulation of somatic
stem cells [25–28]. Downregulation of piwi-like genes in flatworms
results in loss of tissue homeostasis and regeneration capacity,
which finally leads to death [25–26]. These observations suggest a
crucial role of piwi-like genes in somatic stem-cell maintenance in
flatworms. Also, in the acoel I. pulchra, we were able to show the
extended Ipiwi1 expression in a subpopulation of somatic stem
cells, suggesting a similar regulation of both acoel and rhabdito-
phoran stem-cell systems. Furthermore, consistent with the
conspicuous absence of proliferating cells in the epidermis, the
flatworm stem cell marker piwi is not expressed in the epidermal
layer [25,26]. Accordingly, we demonstrate that in the rhabdito-
phoran M. lignano, and in the acoel I. pulchra, piwi-like genes are
expressed in gonads and somatic stem cells, but not in the
epidermis (Fig. 5).
Alternative 1: The stem-cell system is a synapomorphy of
Acoela and Rhabditophora
The new developmental data attest to a possible sister-group
relationship between Acoela and Rhabditophora (Fig. 6A), thereby
contradicting the molecular-phylogeny-based separation of the
acoel species from other flatworms. Neoblast stem cells are located
in the mesodermal space of acoels  (Fig. 2) and rhabditophor-
ans [16,18,20] (Fig. 3), but are absent in the epidermis. In a
previously studied acoel, Convolutriloba longifissura, it is less apparent
that proliferating cells are missing in the epidermis, due to insunk
epidermal nuclei .
Morphological characteristics (i.e. the distribution of stem cells
and the peculiar mode of epidermal replacement) and gene-
expression patterns of the stem-cell marker piwi both confirm the
unique mode of epidermal replacement by mesodermally located
stem cells in acoels and rhabditophorans. This is the first
experimental evidence for complex and robust synapomorphic
characters of the Acoela and Rhabditophora. Hence, the inclusion
of Acoela in the Platyhelminthes can be seen as the most
parsimonious explanation for the presence of epidermal cell
renewal from mesodermally located stem cells and for the piwi-like
gene expression in adult somatic stem cells (Fig. 6A). However, the
peculiar mode of epidermal cell renewal exclusively from
mesodermally located stem cells may not be a synapomorphy for
all traditional taxa of the Platyhelminthes, i.e. the Catenulida, the
Acoela, the Nemertodermatida and the Rhabditophora . Stem
cells in catenulids have long been recognized as being different
from those of other flatworms in that they appear in the epidermis
[30,31]. Preliminary data indicate that in some (but not all)
Nemertodermatida proliferating cells occur also in the epidermal
layer . To justify a monophyletic Platyhelminthes, this
PLoS ONE | www.plosone.org2May 2009 | Volume 4 | Issue 5 | e5502
Figure 1. Phylogenetic analysis of 23 animal species using partial sequences of 43 genes. The acoel Isodiametra pulchra appears as a
sister group of the rest of the bilaterians, and not as a member of the Platyhelminthes. Numbers above nodes refer to the maximum likelihood
boostraps. Values below nodes represent bootstrap support under CAT. Circled numbers indicate the percentage of individual-loci trees that
supported the respective node in the maximum-likelihood analyses of each data-set separately.
PLoS ONE | www.plosone.org3 May 2009 | Volume 4 | Issue 5 | e5502
character could be regarded as being secondarily derived in the
Nemertodermatida and, probably independently, also in the
Interestingly, two previous multi-gene phylogenies show some
support for the grouping of acoels with other flatworms: ‘‘the
standard WAG model groups together the two long branches of
Platyhelminthes and the acoel’’ , and the supplementary
figures 2 and 3 in  feature trees (calculated with maximum
parsimony and the WAG model, respectively), where the acoels
appear basal to rhabditophoran flatworms. The authors of these
papers reject these phylogenies on the basis that the new CAT
model is more likely to avoid analytical errors than the previously
widely employed WAG model [13,14].
A number of phylogenetic studies based on single molecules
have suggested a more basal position of Acoela and Nemerto-
dermatida [5–11], while the monophyly of the Catenulida and the
Figure 2. Cell proliferation and cell migration in acoel flatworms. Localization of BrdU-containing cells in the acoels Isodiametra pulchra,
Neochildia fusca and Aphanostoma sp. after a short BrdU pulse (A–B, E–F, H) and 10 days chase (C–D, G). (A, C, E, G–H) wholemounts of adult animals,
(B, D, F) semithin cross sections. Insets: details of the epidermis, encompassed by dotted lines. Anterior to the left. (A–B, E–F, H) Note the lack of S-
phase cells (brown nuclei) in the epidermis after 30 min BrdU pulse. (C–D, G) BrdU-labeled cells (brown nuclei) migrated from the mesodermal space
to the epidermis and differentiated into epidermal cells during the 10 days chase period. Asterisk denotes diatom in digestive parenchyma. Scale bar
is 50 mm for (B, D, F, H), 100 mm for (A, C), and 200 mm for (E, G).
PLoS ONE | www.plosone.org4 May 2009 | Volume 4 | Issue 5 | e5502
Rhabditophora has been recently confirmed . These phylo-
genetic hypotheses are difficult to reconcile with the stem-cell
system being a synapomorphy between acoel and rhabditophoran
flatworms. Yet, phylogenomic approaches have recently chal-
lenged the basal bilaterian position of acoel (and nemertodermatid)
flatworms [12–13]. Given the current state of knowledge, it is
possible that the similar stem-cell systems in Acoela and
Rhabditophora are plesiomorphic or convergent characters.
On the other hand, totipotency of mesodermally located
neoblasts in adult animals could be a character uniting all
Platyhelminthes, but so far has only been experimentally tested in
the Rhabditophora [34,35]. For triclad and macrostomorphan
flatworms it has been shown that neoblasts are responsible for
growth, tissue maintenance, and regeneration of all tissues,
including gonads [16,36–38]. Future studies on the totipotency
of stem cells of acoels, nemertodermatids and catenulids will be
necessary to confirm or rule out this synapomorphy for all
traditional flatworm taxa.
Alternative 2: The stem-cell system of Acoela and
Rhabditophora is a plesiomorphy
If the similarity of the complex stem-cell system shared between
acoels and rhabditophorans was to be explained as a plesiomorphy
derived from a hypothetical urbilaterian (Fig. 6B), and the acoels
are considered as basal bilaterians as supported by our phylogeny
(Fig. 1), at least the following groups would have to have lost this
particular feature: the Deuterostomia, the Ecdysozoa, and the
Eutrochozoa (Annelida and Mollusca) (Fig. 1). Other trees
featuring more taxa (e.g. ) and where acoels hold a different
position imply that the particular mode of epidermal cell renewal
would even have been lost several more times.
Diploblasts such as cnidarians lack a mesodermal layer, making a
direct comparison with the mesodermally located stem cells in the
triploblastic acoel and rhabditophoran flatworms difficult. More-
over, recent phylogenetic studies suggest a placozoan-like ancestor
of bilaterians instead of a cnidarian (planula)-like one .
Epidermal cells are proliferating at least in some cnidarians, and
interstitial cells (i-cells) are found and are proliferating in the
epidermis between epidermal cells. This is in stark contrast to the
neoblastsof acoel andrhabditophoran flatworms, whichareentirely
lacking in the epidermis. Still, considering the presence of totipotent
i-cells in the cnidarian Hydractinia , homology of these i-cells and
the neoblasts in acoels seems possible, but requires multiple gains of
epidermally located stem cells in the Bilateria. In this scenario, the
stem-cell system in both acoels and rhabditophorans constitutes a
plesiomorphy (Fig. 6B), but the same number of gains of stem cells
intheepidermisamongmajorbilaterian taxa isnecessary even if the
stem-cell system is regarded as a plesiomorphy for acoels, and an
apomorphy for rhabditophorans (Fig. 6C).
Alternative 3: The stem-cell system of Acoela and
Rhabditophora is a product of convergent evolution
Regardless of whether the neoblast stem-cell system is a
plesiomorphy or an apomorphy for acoels, the assumption of an
independent development of a very similar stem-cell system in
rhabditophorans indicates a similar need in these two taxa for such
a peculiar stem-cell system.
Only for the Neodermata (parasitic rhabditophoran flatworms,
including tapeworms and flukes), the lack of stem cells within the
epidermis can be seen as a prerequisite for avoiding the host
defense mechanisms by shedding the ciliated epidermis. During
postembryonic development, these parasitic rhabditophoran
flatworms completely replace their primary epidermis with a
Figure 3. Cell proliferation and cell migration in rhabdito-
phoran flatworms. Localization of BrdU-containing cells in the
rhabditophorans Prosthiostomum siphunculus, Pseudostylochus interme-
dius, Planocera reticulata (A–C, Polycladida) and Macrostomum spirale
(D, Macrostomorpha). (A) Anterior part of an adult, (B–D) juveniles.
Insets: details of the epidermis, encompassed by dotted lines. Anterior
to the left. (A–B, D) Note the lack of S-phase cells (brown or green
nuclei) in the epidermis after 30 min or 12 h (in B) BrdU pulse. (A) Lower
inset shows the protruding pharynx also lacking proliferating cells. (C)
BrdU-labeled cells (green nuclei) migrated from the mesodermal space
to the epidermis and differentiated into epidermal cells during the 7
days chase period. Scale bar is 50 mm for (D), 100 mm for (B), 400 mm for
(C) and 1 mm for (A).
PLoS ONE | www.plosone.org5 May 2009 | Volume 4 | Issue 5 | e5502
newly formed syncytial epidermal layer derived from mesoder-
mally located stem cells . Some acoels and rhabditophorans
share a thin epidermis and weak basal matrix, which might be
related to the loss of an intra-epidermal stem-cell system. Many
flatworms also share a similar habitat, the mesopsammon , but
other representatives of the interstitial fauna, such as annelids and
nemerteans do have proliferating stem cells in the epidermis
(Fig. 4), showing that the acoel and rhabditophoran stem-cell
system is not a necessity to survive in this habitat.
Considering the obvious conflict between molecular phylogenies
and morphological data, the monophyly of the flatworms remains
undecided. Although molecular phylogenies show a position of the
Acoela separate from the remaining flatworms, the stem-cell
system provides two strong synapomorphies for the Rhabdito-
phora and the Acoela: 1) epidermal replacement exclusively
through mesodermally located stem cells, and 2) expression of a
piwi-like gene also in somatic, not only in gonadal stem cells. The
alternative would be that the highly similar stem-cell system
evolved in parallel in Acoela and Rhabditophora, or is a
plesiomorphic feature that was retained.
Recently, the myxozoan worm Buddenbrockia has been identified
as a member of the Cnidaria by molecular means despite striking
morphological dissimilarity . While this conflict between
morphological and molecular characters can be readily accounted
for by the morphological reductionism resulting from the parasitic
lifestyle of Buddenbrockia, no such accounting can explain the suite
of morphological characters shared among Platyhelminthes . In
particular, the special mode of epidermal replacement in acoels
and rhabditophorans constitutes an apomorphy supporting a
possible sister-group relationship between these taxa. The
available multi-locus phylogenies, which largely do not even agree
with one another concerning the placement of acoels, cannot
Figure 4. Cell proliferation in other spiralians: an annelid and a nemertean. Localization of BrdU-containing cells in the annelid Dorvillea
bermudensis (A–C) and the nemertean Cephalothrix sp. (D–E) after 30 min incubation of BrdU. (A) Wholemount of the posterior segments of D.
bermudensis. (B) Semithin cross section through midbody and parapodia of animal shown in (A). Labeled cells are located in the epidermis, the
mesodermal space, and the gastrodermis (indicated by asterisk). (C) Magnified view of labeled epidermal cells shown in (B). (D) Anterior end of
Cephalothrix sp. Labeled cells in the epidermis are separated from muscular layers and the cutis by a light-brown basal matrix. Labeled cells are also
present in the mesodermal space. Inset shows details of labeled epidermal cells. (E) More posterior part of the animal than (D) with labeled cells in the
epidermis. Arrowheads denote the proboscis. Scale bar is 100 mm for (A), 20 mm for (B), 5 mm for (C), and 50 mm for (D–E).
PLoS ONE | www.plosone.org6 May 2009 | Volume 4 | Issue 5 | e5502
resolve the validity of a sister-group relationship between the
Acoela and the Rhabditophora. The remaining taxa of the
traditional Platyhelminthes, the Catenulida and possibly the
Nemertodermatida do not share the peculiar stem cell system of
Acoela and Rhabditophora and may lie at the base of the
flatworms, may have secondarily evolved proliferating stem cells in
the epidermis, or may not be flatworms at all. It appears that until
substantial sampling of lower taxa among flatworms is performed,
and more studies on stem cells in non-rhabditophoran flatworms
are available, none of the competing phylogenetic hypotheses can
be favored. Therefore, we concur with Tor Karling  that
‘‘…the search for sister groups throws a sharp light on our
insufficient knowledge of the phylogenetic connections [among]
the turbellarian taxa….’’
Materials and Methods
BrdU labeling was performed according to  except for using
2.5% glutaraldehyde in 0.1 M cacodylate buffer and 9% sucrose
for fixation, a StreptABComplex/HRP Duet kit (DAKO) for
secondary antibodies and visualization for precipitation of the
BrdU label (brown label). Also, different times of BrdU (12 hours
pulse instead of 30 min for juvenile of Pseudostylochus intermedius) and
protease incubation were used for different species, and treatment
with 0.1 M HCl after protease incubation was omitted.
In situ hybridization
Whole mount in situ hybridization (ISH) on M. lignano was
carried out as described previously . For I. pulchra, the same
protocol was used, except for Proteinase K treatment, which was
applied for 7 min only. Sense and antisense riboprobes were
generated using the DIG RNA labeling KIT SP6/T7 (Roche),
following the manufacturer’s protocol. During hybridization,
riboprobes were used at a final concentration of 0.05 ng/ml.
The following primer couples were used for generating in situ
TGTTGCTGGTC-39 and 59-GTCTTGTTGTTGTGCCGC-
GTGAG-39. For Ipiwi1 59-CATGCTGGAGATGGGCAAGAT-
Partial sequences of piwi-like genes were obtained from the
Macrostomum lignano EST database (Angu7606) (Morris et al. 2006)
and unpublished Isodiametra pulchra ESTs (Contig 447) (Ladurner
and Agata, unpublished). Both gene sequences were submitted to
AM942740). Detailed information of both genes will be published
We used available EST (expressed sequence tags) and whole
genome databases in order to obtain multi-locus data matrices for
phylogenetic inferences (see Table S1). The approach of
combining dozens of homologous gene fragments for phylogenetic
reconstruction has been applied successfully in previous phyloge-
nomic studies and seems particularly useful when, for some taxa of
interest, only limited genomic resources are available. One
advantage of multi-locus phylogenies over single-locus ones is
the increase in robustness, which is essentially due to the much
larger number of phylogenetic informative positions. Furthermore,
an increase in sequence length generally leads to a smaller
variance in evolutionary rates and other parameters in model-
(many) single gene-alignments may effectively correct for the
erroneous phylogenetic signal contained in single genes, and it has
been shown that even genes producing incongruent phylogenies
are useful in multi-gene alignments, as they may provide
additional information for resolving at least some short branches.
On the other hand, the analyses of multi-locus datasets are more
challenging. For example, it is often difficult, if not impossible, to
assign appropriate model parameters for each partition individ-
ually. It has been suggested, though, that the increase in the
phylogenetic signal and/or signal/noise-ratio due to concatenation
has a much stronger effect on the resulting phylogenies than any
bias introduced by averaging over model parameters.
Here, we used as starting point a set of 4,885 ESTs from our
Isodiametra pulchra cDNA-sequencing project (Ladurner and Agata,
unpublished). These sequences were used as query for tblastx
homology searches against all other 23 databases (Table S1). For
these blast searches, we used an e-value #10250to acquire
sequences with a large enough degree of sequence homology to be
suitable for phylogeny reconstruction and to avoid the erroneous
inclusion of paralogs. We then applied EVEREST to assign the
‘‘best hit’’ sequences with respect to the Isodiametra homolog from
every BLAST search. We ended up with a total of 32 loci that
were present in all organisms. When excluding the smallest
database, Macrostomum lignano, for which only 1,231 cDNA
fragments were available, we obtained 43 genes that were
unambiguously present in all remaining taxa and conserved
enough to allow alignment. Homologous protein sequences were
aligned with CLUSTAL X, resulting in two datasets containing 32
and 43 fragments, respectively. GenBank accession numbers of the
analyzed sequences are listed in Table S2, the number of amino
acid positions used for the phylogenetic analyses are listed in Table
S3. Outgroup status was assigned to Ephydatia fluviatilis because
Figure 5. Piwi-like gene expression in an acoel and a
rhabditophoran flatworm. In situ hybridizations of adult animals.
(A) Isodiametra pulchra (Acoela), (B) Macrostomum lignano (Rhabdito-
phora). Expression of piwi-like genes in the germ line and somatic stem
cells. Note the lack of Ipiwi1 (A) and Macpiwi (B) expression in the
epidermis. Insets: details of the epidermis, encompassed by dotted
lines. Accession numbers: Ipiwi1 AM942741, Macpiwi AM942740. Scale
bar is 100 mm.
PLoS ONE | www.plosone.org7 May 2009 | Volume 4 | Issue 5 | e5502
sponges are a valid sister group to the remaining metazoans;
cnidarians are a valid sister group to the bilaterians.
Maximum likelihood and Bayesian analyses were performed
with both datasets and with single loci as well as multi-gene
alignments including all sequence fragments. The total length of
the concatenated dataset including Macrostomum lignano was 6,718
amino acid positions (32 loci), while the concatenated dataset
without Macrostomum lignano (43 loci) had 10,218 amino acid
positions. 4,903 positions of the alignment contained gaps in at
least one of the taxa. Since the resulting missing data represent less
than half of the combined sequence, we included these taxa in the
Figure 6. Alternative hypotheses of evolution of epidermal replacement. (A) Alternative 1: The similar stem-cell system between Acoela and
Rhabditophora is a synapomorphy. This scenario requires a single loss of epidermal stem cells. Notably, the observation of mitotic figures in the
epidermis of catenulids supports a sister group relationship of the Catenulida to the Acoela and Rhabditophora. (B) Alternative 2a: The similar stem-
cell system between Acoela and Rhabditophora is a plesiomorphy in both taxa. This requires the independent gain of stem cells in the epidermis by
the Catenulida, the coelomate Spiralia and other Bilateria not shown in the diagram. (C) Alternative 2b: The similar stem-cell system between Acoela
and Rhabditophora is a plesiomorphy in Acoela and a convergent character in Rhabditophora. This requires the gain of stem cells in the epidermis in
the Spiralia and other Bilateria not shown in the diagram. (D) Alternative 3: The similar stem-cell system between Acoela and Rhabditophora is a
convergent character that was independently developed in both Acoela and Rhabditophora.
PLoS ONE | www.plosone.org8 May 2009 | Volume 4 | Issue 5 | e5502
Prior to phylogenetic analyses, model selections with different
model selection strategies (AIC, AICc, BIC) were performed with
PROTTEST with all single-gene and multi-gene alignment files.
According to the results obtained, we performed maximum-
likelihood analysis and100 maximum-likelihood bootstrap
replicates with PHYML applying the WAG+C model (gamma
shape parameter a=0.77) of sequence evolution for the
concatenated file including 32 loci, and the WAG+I+C model
(gamma shape parameter a=1.29, proportion of invariant sites
0.06) for the multi-gene dataset containing 43 gene fragments.
We also applied a mixed-model approach to both datasets using
CAT, a previously developed model accounting for site-specific
amino acid replacement patterns . To avoid local minimum in
tree space search (especially the artefactual attraction of nema-
todes and platyhelminths, see , we used two different starting
trees (the most parsimonious one and the one obtained by CAT)
and retained the tree with the highest likelihood. Bayesian
phylogenetic analyses under the CAT model were performed
using the PhyloBayes package (www.lirmm.fr/mab, ). For the
plain posterior estimation, four independent chains were run for a
total number of 15,000 cycles, saving every cycle, and discarding
the first 1,500 cycles (burn-in). In all cases, the two independent
experiments always lead to the same tree. Therefore, the posterior
consensus tree was obtained by pooling both the tree lists of four
independent runs. For both models, we measured clade support by
non-parametric bootstrap with 100 replicates. To reduce compu-
tational burden for the CAT model, a run of 4,000 cycles,
discarding the first 1000 as burn-in was performed. The posterior
consensus tree was computed for each replicate, and the majority-
rule consensus of these 100 trees was our final bootstrap estimate.
In order to test the phylogenetic signal and the contribution of
each of the single loci to the general topology, we calculated the
number of single-gene trees supporting a given partition of the
general topology. To this end, maximum-likelihood trees were
constructed from each single-gene alignment with PHYML,
applying the model of sequence evolution and the respective
parameters according to the model selection with PROTTEST. The
percentage fraction of single-gene trees containing a particular node
is depicted on the branches in Fig. 1. With this approach, and the
stringent search criteria in the blast searches, we could exclude the
possibility that our alignment files included paralogous genes.
Alternative topologies constraining monophyly of the Platyhel-
minthes were compared applying the approximately unbiased
(AU) test as implemented in the CONSEL package, using the
sidewise likelihood values estimated by PAML. For both datasets,
the AU test revealed that the maximum likelihood phylogenies
placing Isodiametra pulchra as most ancestral taxon, sister group to
all remaining bilaterians (Fig. 1, Fig S1), was significantly better
based on the available data (p.0.01) compared to trees in which
the monophyly of the Platyhelminthes was enforced (Table S4).
analysis of 24 species using partial sequences of 32 genes. The acoel I.
pulchra appears as a sister group of the rest of the bilaterians, and not
as a member of the platyhelminthes. The macrostomorphan M.
lignano lies basal to other rhabditophoran flatworms (Tricladida,
Neodermata). Numbers above nodes refer to the maximum
likelihood boostraps.Values below nodes represent bootstrap support
under CAT. Circled numbers indicate the percentage of individual-
loci trees that supported the respective node in the maximum-
likelihood analyses of each data-set separately.
Found at: doi:10.1371/journal.pone.0005502.s001 (0.82 MB TIF)
Tree of phylogenetic analysis of 24 species. Phylogenetic
Found at: doi:10.1371/journal.pone.0005502.s002 (0.05 MB
Species used for the phylogenetic tree reconstruction.
numbers of the sequences used for the phylogenetic analyses.
Found at: doi:10.1371/journal.pone.0005502.s003 (0.19 MB
GenBank accession numbers. GenBank accession
used for the phylogenetic analyses.
Found at: doi:10.1371/journal.pone.0005502.s004 (0.03 MB
Length of the sequences used. Length of the sequences
position of the acoel Isodiametra pulchra in the phylogenetic tree.
Found at: doi:10.1371/journal.pone.0005502.s005 (0.04 MB
Testing the position of the acoel in the tree. Testing the
Monika Mu ¨ller is to be thanked for providing specimens of Dorvillea
bermudensis. We also thank Raju Tomer for his help with Platynereis sequence
submissions. Part of this study was performed at the Gene Research Center
of Hirosaki University.
Conceived and designed the experiments: BE DS HT KDM RR ST KA
WS PL. Performed the experiments: BE DS HT KDM RG MH CK GK
ON DP WS PL. Analyzed the data: BE DS HT KDM DA GB NF RG VH
BH MH MH SI CK GK ON DP RR WS JSI UT ST KA WS PL.
Contributed reagents/materials/analysis tools: BE DS HT DA GB NF RG
VH BH MH SI JSI UT ST KA WS PL. Wrote the paper: BE DS KDM
RR JSI ST KA WS PL.
1. Karling TG (1974) On the anatomy and affinities of the turbellarian orders. In:
Riser NW, Morse MP, eds. Biology of the Turbellaria. New York: McGraw Hill.
2. Ehlers U (1985) Das Phylogenetische System der Plathelminthes. Stuttgart:
Fischer. 317 p.
3. Tyler S, Hooge M (2004) Comparative morphology of the body wall in
flatworms (Platyhelminthes). Can J Zool 82: 194–210.
4. Smith JPS, Tyler S, Rieger RM (1986) Is the Turbellaria polyphyletic?
Hydrobiologia 132: 13–2.
5. Ruiz-Trillo I, Riutort M, Littlewood DT, Herniou EA, Bagun ˜a ` J (1999) Acoel
flatworms: earliest extant bilaterian Metazoans, not members of Platyhelmin-
thes. Science 283: 1919–1923.
6. Ruiz-Trillo I, Paps J, Loukota M, Ribera C, Jondelius U, et al. (2002) A
phylogenetic analysis of myosin heavy chain type II sequences corroborates that
Acoela and Nemertodermatida are basal bilaterians. Proc Natl Acad Sci U S A
7. Ruiz-Trillo I, Riutort M, Fourcade HM, Bagun ˜a ` J, Boore JL (2004)
Mitochondrial genome data support the basal position of Acoelomorpha and
the polyphyly of the Platyhelminthes. Mol Phylogenet Evol 33: 321–332.
8. Telford MJ, Lockyer AE, Cartwright-Finch C, Littlewood DT (2003) Combined
large and small subunit ribosomal RNA phylogenies support a basal position of
the acoelomorph flatworms. Proc R Soc Lond B Biol Sci 270: 1077–1083.
9. Bagun ˜a ` J, Riutort M (2004) The dawn of bilaterian animals: the case of
acoelomorph flatworms. Bioessays 26: 1046–1057.
10. Jimenez-Guri E, Paps J, Garcia-Fernandez J, Salo E (2006) Hox and ParaHox
genes in Nemertodermatida, a basal bilaterian clade. Int J Dev Biol 50: 675–679.
11. Sempere LF, Martinez P, Cole C, Bagun ˜a ` J, Peterson KJ (2007) Phylogenetic
distribution of microRNAs supports the basal position of acoel flatworms and the
polyphyly of Platyhelminthes. Evol Dev 9: 409–415.
12. Dunn CW, Hejnol A, Matus DQ, Pang K, Browne WE, et al. (2008) Broad
phylogenomic sampling improves resolution of the animal tree of life. Nature
PLoS ONE | www.plosone.org9 May 2009 | Volume 4 | Issue 5 | e5502
13. Philippe H, Brinkmann H, Martinez P, Riutort M, Bagun ˜a ` J (2007) Acoel Download full-text
flatworms are not Platyhelminthes: evidence from phylogenomics. PLoS ONE 2:
14. Bagun ˜a ` J, Martinez P, Paps J, Riutort M (2008) Back in time: a new systematic
proposal for the Bilateria. Phil Trans R Soc B 363: 1481–1491.
15. Jimenez-Guri E, Philippe H, Okamura B, Holland PWH (2007) Buddenbrockia is a
cnidarian worm. Science 317: 116–118.
16. Ladurner P, Rieger R, Bagun ˜a ` J (2000) Spatial distribution and differentiation
potential of stem cells in hatchlings and adults in the marine platyhelminth
Macrostomum sp.: A Bromodeoxyuridine analysis. Dev Biol 226: 231–241.
17. Drobysheva IM (1988) Drobysheva IM (1988) An autoradiographic study of the
replacement of epidermis in polyclad turbellarians. Fortschr Zool 36: 97–101.
18. Newmark PA, Sa ´nchez Alvarado A (2000) Bromodeoxyuridine specifically labels
the regenerative stem cells of planarians. Dev Biol 220: 142–153.
19. MacKinnon BM, Burt MDB, Pike AW (1981) Ultrastructure of the epidermis of
adult and embryonic Paravortex species (Turbellaria, Eulecithophora). Hydro-
biologia 84: 241–252.
20. Gustafsson MK (1976) Studies on cytodifferentiation in the neck region of
Diphyllobothrium dendriticum Nitzsch, 1824 (Cestoda, Pseudophyllidea).
Z Parasitenkd 50: 323–329.
21. Paulus T, Mu ¨ller MCM (2006) Cell proliferation dynamics and morphological
differentiation during regeneration in Dorvillea bermudensis (Polychaeta, Dorvillei-
dae). J Morphol 267: 393–403.
22. Hanselmann R, Smolowitz R (2000) Identification of proliferating cells in hard
clams. Biol Bull 199: 199–200.
23. O’Donnell KA, Boeke JD (2007) Mighty Piwis defend the germline against
genome intruders. Cell 129: 37–44.
24. Klattenhoff C, Theurkauf W (2008) Biogenesis and germline functions of
piRNAs. Development 135: 3–9.
25. Reddien PW, Oviedo NJ, Jennings JR, Jenkin JC, Sa ´nchez Alvarado A (2005)
SMEDWI-2 is a PIWI-like protein that regulates planarian stem cells. Science
26. Palakodeti D, Smielewska M, Lu YC, Yeo GW, Graveley BR (2008) The PIWI
proteins SMEDWI-2 and SMEDWI-3 are required for stem cell function and
piRNA expression in planarians. RNA 14: 1174–1186.
27. Guo T, Peters AH, Newmark PA (2006) A Bruno-like gene is required for stem
cell maintenance in planarians. Dev Cell 11: 159–169.
28. Rossi L, Salvetti A, Lena A, Batistoni R, Deri P, et al. (2006) DjPiwi-1, a
member of the PAZ-Piwi gene family, defines a subpopulation of planarian stem
cells. Dev Genes Evol 216: 335–346.
29. Gschwentner R, Ladurner P, Nimeth K, Rieger R (2001) Stem cells in a basal
bilaterian: S-phase and mitotic cells in Convolutriloba longifissura (Acoela,
Platyhelminthes). Cell Tissue Res 304: 401–408.
30. Ott HN (1892) A study of Stenostoma leucops O Schm. J Morphol 7: 263–304.
31. Ehlers U (1992) No mitosis of differentiated epidermal cells in the
Plathelminthes: mitosis of intraepidermal stem cells in Rhynchoscolex simplex
Leidy, 1851 (Catenulida). Microfauna Marina 7: 311–321.
32. Smith III JPS, Egger B, Tyler S, Ladurner P, Achatz J, et al. (2009) Neoblasts in
Nemertodermatida. Soc Integ Comp Biol Meeting Abstract, http://www.sicb.
33. Larsson K, Jondelius U (2008) Phylogeny of Catenulida and support for
Platyhelminthes. Org Divers Evol 8: 378–387.
34. Bagun ˜a ` J, Salo E, Auladell C (1989) Regeneration and pattern formation in
planarians III. Evidence that neoblasts are totipotent stem cells and the source of
blastema cells. Development 107: 77–86.
35. Toledo A, Cruz C, Fragoso G, Laclette JP, Merchant MT, et al. (1997) In vitro
culture of Taenia crassiceps larval cells and cyst regeneration after injection into
mice. J Parasitol 83: 189–193.
36. Bode A, Salvenmoser W, Nimeth K, Mahlknecht M, Adamski Z, et al. (2006)
Immunogold-labeled S-phase neoblasts, total neoblast number, their distribu-
tion, and evidence for arrested neoblasts in Macrostomum lignano (Platyhelminthes,
Rhabditophora). Cell Tissue Res 325: 577–587.
37. Egger B, Ladurner P, Nimeth K, Gschwentner R, Rieger R (2006) The
regeneration capacity of the flatworm Macrostomum lignano - on repeated
regeneration, rejuvenation, and the minimal size needed for regeneration. Dev
Genes Evol 216: 565–577.
38. Reddien PW, Sa ´nchez Alvarado A (2004) Fundamentals of planarian
regeneration. Annu Rev Cell Dev Biol 20: 725–757.
39. Dellaporta SL, Xu A, Sagasser S, Jakob W, Moreno MA, et al. (2006)
Mitochondrial genome of Trichoplax adhaerens supports Placozoa as the basal
lower metazoan phylum P. Natl Acad Sci USA 103: 8751–8756.
40. Mu ¨ller WA, Teo R, Frank U (2004) Totipotent migratory stem cells in a
hydroid. Dev Biol 275: 215–224.
41. Tyler S, Hooge M (2004) Comparative morphology of the body wall in
flatworms (Platyhelminthes). Can J Zool 82: 194–210.
42. Rieger R (2006) Plathelminthes, Plattwu ¨rmer. In: Westheide W, Rieger R, eds.
Spezielle Zoologie I. Einzeller und Wirbellose Tiere. Stuttgart: Gustav Fischer
Verlag. pp 209–260.
43. Pfister D, De Mulder K, Philipp I, Kuales G, Hrouda M, et al. (2007) The
exceptional stem cell system of Macrostomum lignano: screening for gene expression
and studying cell proliferation by hydroxyurea treatment and irradiation. Front
Zool 4: 9.
44. Lartillot N, Philippe H (2004) A Bayesian mixture model for across-site
heterogeneities in the amino-acid replacement process. Mol Biol Evol 21:
45. Philippe H, Lartillot N, Brinkmann H (2005) Multigene analyses of bilaterian
animals corroborate the monophyly of Ecdysozoa, Lophotrochozoa, and
Protostomia. Mol Biol Evol 22: 1246–53.
46. Lartillot N, Brinkmann H, Philippe H (2007) Suppression of long-branch
attraction artefacts in the animal phylogeny using a site-heterogeneous model.
BMC Evol Biol 7(Suppl 1): S4.
PLoS ONE | www.plosone.org10 May 2009 | Volume 4 | Issue 5 | e5502