EST sequencing of Onychophora and phylogenomic analysis of Metazoa
Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873 5' cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs. BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than 10(-4). Most ESTs had the best hit with proteins from either Chordata or Arthropoda (approximately 40% respectively). The ESTs included sequences of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases, including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 positions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved. The "Articulata" concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardigrada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.
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EST sequencing of Onychophora and phylogenomic analysis
, Silke Hagner-Holler
, Hilke Ruhberg
, Ingo Ebersberger
Arndt von Haeseler
, Michael Kube
, Richard Reinhardt
, Thorsten Burmester
Institute of Zoology and Zoological Museum, University of Hamburg, D-20146 Hamburg, Germany
Institute of Zoology, University of Mainz, D-55099 Mainz, Germany
Center for Integrative Bioinformatics in Vienna, Max F. Perutz Laboratories, University of Vienna, Medical University Vienna,
University of Veterinary Medicine Vienna, A-1030 Vienna, Austria
Max Planck Institute for Molecular Genetics, Ihnestrasse 63, D-14195 Berlin, Germany
Received 13 February 2007; revised 29 August 2007; accepted 5 September 2007
Available online 12 September 2007
Onychophora (velvet worms) represent a small animal taxon considered to be related to Euarthropoda. We have obtained 1873
cDNA sequences (expressed sequence tags, ESTs) from the velvet worm Epiperipatus sp., which were assembled into 833 contigs.
BLAST similarity searches revealed that 51.9% of the contigs had matches in the protein databases with expectation values lower than
. Most ESTs had the best hit with proteins from either Chordata or Arthropoda (40% respectively). The ESTs included sequences
of 27 ribosomal proteins. The orthologous sequences from 28 other species of a broad range of phyla were obtained from the databases,
including other EST projects. A concatenated amino acid alignment comprising 5021 positions was constructed, which covers 4259 posi-
tions when problematic regions were removed. Bayesian and maximum likelihood methods place Epiperipatus within the monophyletic
Ecdysozoa (Onychophora, Arthropoda, Tardigrada and Nematoda), but its exact relation to the Euarthropoda remained unresolved.
The ‘‘Articulata’’ concept was not supported. Tardigrada and Nematoda formed a well-supported monophylum, suggesting that Tardi-
grada are actually Cycloneuralia. In agreement with previous studies, we have demonstrated that random sequencing of cDNAs results
in sequence information suitable for phylogenomic approaches to resolve metazoan relationships.
Ó 2007 Elsevier Inc. All rights reserved.
Keywords: Arthropoda; Onychophora; Ecdysozoa; EST
The phylum Onychophora (velvet worms) includes
about 180 described species (Ruhberg, 2007). These soft-
bodied, terrestrial animals with caterpillar-like appearance
and size are restricted to the Southern Hemisphere, and to
tropical and subtropical regions in the Northern Hemi-
sphere. Onychophora are classiﬁed into two families, Peri-
patidae and Peripatopsidae (Ruhberg, 1985).
Onychophoran-like lobopodian fossils have been found in
Lower and M iddle Cambrian strata, suggesting an early
evolutionary origin of this taxon (Hou and Bergstro
1995). In the ﬁrst taxonomic description, Onychophora
were considered as modiﬁed molluscs (Guilding, 1826),
but today they are treated either as separate invertebrate
phylum or as a subphylum of the (Pan-)Arthropoda (Niel-
sen, 2001; Brusca and Brusca, 2003). In any case, molecular
phylogenetic studies agree with that Onychophora are clo-
sely allied with the (Eu-)Arthropoda (Ballard et al., 1992;
Boore et al., 1995; Edgecombe et al., 2000; Giribet et al.,
2001; Kusche et al., 2002).
Onychophora and Euarthropoda are members of the
superphylum Ecdysozoa, which also includes nematodes
and allied phyla (‘‘Cycloneuralia’’). This classiﬁcation
was originally proposed by Aguinaldo et al. (1997) on the
1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved.
Corresponding author. Fax: +49 40 42838 3937.
E-mail address: email@example.com (T. Burmester).
Available online at www.sciencedirect.com
Molecular Phylogenetics and Evolution 45 (2007) 942–951
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basis of phylogenetic analysis of 18S rRNA sequences. In
addition to the rRNA data (Aguinaldo et al., 1997; Mallatt
et al., 2004), the Ecdysozoa concept is supported by the
analyses of HOX genes, myosin heavy chain II and
sodium-potassium ATPase sequences (de Rosa et al.,
1999; Ruiz-Trillo et al., 2002; Anderson et al., 2004;
Kusche et al., 2005), by mitochondrial gene arrangements
(Lavrov and Lang, 2005), as well as by rare amino acid
replacements (Irimia et al., 2007). Morphological evidence
in favour for the Ecdysozoa concept exists but is still scarce
(cf. Schmidt-Rhaesa et al., 1998; Haase et al., 2001; Peter-
son and Eernisse, 2001; Giribet, 2003). Other molecular
phylogenetic studies supported the traditional view, which
places the Nematoda as ‘‘Pseudocoelomata’’ as sistergroup
of the ‘‘Coelomata’’ (Hausdorf, 2000; Blair et al., 2002;
Dopazo et al., 2004; Wolf et al., 2004; Philip et al., 2005;
Rogozin et al., 2007).
Usually, metazoan phylogeny has been investigated on
the basis of single genes (e.g., Aguinaldo et al., 1997; de
Rosa et al., 1999; Ruiz-Trillo et al., 2002; Anderson
et al., 2004; Mallatt et al., 2004; Kusche et al., 2005)
or by supergene analyses of speciﬁcally selected genes
(e.g., Wolf et al., 2004). An alternative approach is the
usage of sequences obtained from ‘‘Expressed Sequence
Tags’’ (ESTs). ESTs are partial cDNA sequences that
derive from single-pas s sequencing (Adams et al., 1991;
Boguski et al., 1993). They provide a rich source of
information that has been used for the identiﬁcation of
novel genes, gene mapp ing, comparative genomics and
functional characteriza tion of gene products (e.g., Rudd,
2003). The application of ESTs in reconstruction of phy-
logenetic relationships is a rather recent development. In
this approach, gene fragments are obtaine d via sequenc-
ing of randomly selected cDNA-clones. Overlapping
sequences are clustered, and orthol ogs to known genes
are determined by means of sequence similarity. These
sequences are then employed to construct the phyloge-
netic trees. Bapteste et al. (2002) used ESTs to study
the phylogenetic aﬃnities of amoebans and related taxa.
Scholl and Bird (2005) investigated the relationships
among some plant-parasitic nematodes based on an
EST-derived concatenated alignment. Steinke et al.
(2006) employed EST data to investigate the phylogeny
of teleost ﬁsh, and Hughes et al. (2006) studied the
molecular systematic of Coleoptera via EST sampling.
Philippe et al. (2004, 2005) used large EST-alignments
to derive the phylogeny within and among Metazoa
and related eukaryotic taxa. Most recently, Bourlat
et al. (2006) used EST sequencing to place the enigmatic
taxon Xenoturbella reliably within the Deuterostomia.
These studies oﬀer promising perspectives that ESTs
contain suﬃcient phylogenet ic information to resolve deep
metazoan phylogeny (Philippe and Telford, 2006). In this
study, we have obtained 1873 ESTs from an ampliﬁed
cDNA library of the onychophoran species Epiperipatus
sp. and used ribosomal proteins to infer the phylogeny of
2. Materials and methods
Epiperipatus sp. (Onychophora, Peripatidae) was origi-
nally captured March, 3 1999 in Nuevo Arenal, Costa
W, 640 m NN, by W . Boeckeler
and I. Richling. Specimens used in this study were bred in
the laboratory by H. Ruhberg (Hamburg).
2.2. RNA extraction and cDNA library construction
Total RNA was extracted from two adult specimens using
the guanidine-thiocyanate method (Chirgwin et al., 1979).
Two specimens of Epiperipatus sp. were pulverized in liquid
nitrogen and about 1 ml per 100 mg tissue guanidine isothi-
ocyanate solution (100 g guanidine isothiocyanate in 117 ml
O, 7.0 ml of 0.75 M sodium citrate, pH 7.0, 10.6 ml 10%
sarcosyl, 1.52 ml b-mercaptoethanol). The solution was sup-
plemented with 1 volume phenol, saturated with 50 mM
Tris–HCl, pH 4.0, and 0.1 volumes chloroform/isoamylalco-
hol, and incubated for 15 min on ice. After centrifugation for
45 min at 13,000g and 4 °C, the RNA was obtained from the
aqueous supernatant by precipitation with 1 volume ethanol.
The RNA pellet was dissolved in diethylpyrocarbonate-trea-
O for further use. Poly(A)
RNA was puriﬁed from
the total RNA using the PolyATract
kit (Promega). About
3 lg poly(A)
RNA was used for the construction of a direc-
tionally cloned cDNA library. The Lambda Zap Express
cDNA Synthesis Kit (Stratagene) was employed for the con-
version of the mRNA into cDNA and the Gigapack III Gold
Cloning Kit (Stratagene) to create the phage library accord-
ing to the manufa cturer’s instructions. Phage was converted
to pBK-CMV plasmids by mass in vivo excision using mate-
rials and protocols provided by Stratagene.
2.3. EST sequencing and clustering
Using standard EST protocols, individual clones were
isolated, the inserts were ampliﬁed by PCR and sequences
were obtained from the 5
end with BigDye chemi stry
(ABI). Collected reads from 3730XL capillary sequencers
(ABI) were base-called using Phred (http://www.phra-
p.org) and subsequently quality and vector clipped using
Lucy (http://compbio.dfci.harvard.edu/tgi/software) with
standard parameters. An average sequence length of 429
bases was obtained after clipping. For clustering and
assembly, the TGI tools developed at TIGR (http://comp-
bio.dfci.harvard.edu/tgi/software) were used. Epiperipatus
sp. ESTs have been deposited in the GenBank, EMBL,
and DDBJ nucleotide sequence databases under the Acces-
sion Nos. AM498769 to AM500636.
2.4. Sequence analyses
Epiperipatus ESTs were compared with the NCBI pro-
tein database using the Bla stX algorithm as implemented
F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951 943
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in the Blastcl3 client available from the National Center for
Biotechnology Information (http://www.ncbi.nlm.nih.
gov). Expectation (E-) values of 10
applied as cut-oﬀs in the BlastX analyses. Sequences of
obviously mitochondrial origin were excluded from further
studies. Gene ontology was predicted by a semi-automatic
gene annotator (GOﬁgure, http://udgenome.ags.udel.edu/
frm_go.html). The number of functional classes was limited
according to Harcus et al. (2004).
2.5. Metazoan ribosomal proteins
The amino acid sequences of Drosophila melanogaster
and Homo sapiens ribosomal proteins were retrieved from
public databases. Data from the two species were com-
bined into a single data ﬁle and were employed to search
the genome and EST sequences of selected metazoan spe-
cies via TBlastN. Matching ESTs that harboured suﬃcient
coverage of ribosomal proteins (P50%) were analyzed fur-
ther (cf. Table 1). For the individual species and the indi-
vidual ribosom al proteins we assembled overlapping
ESTs with the Vector NTI 10 program (Invitrogen). The
resulting contigs were checked for assembly errors by
visual inspection and by comparison with orthologous
sequences of related taxa. Protein sequences were obtained
by the ExPASy Translate tool (http://www.expasy.org/
2.6. Multiple sequence alignments
The deduced protein sequences of each ribosomal protein
were aligned by ClustalX (Thompson et al., 1997)(Supple-
mental Fig. 1). The alignments were visually inspected and
adjusted by hand using GeneDoc 2.6 (Nicholas and Nicho-
las, 1997). Then, the individual alignments were concate-
nated to form a single concatenated multiple sequence
alignment. Gblocks (Castresana, 2000; http://molevol.
ibmb.csic.es/Gblocks_server/index.html) was e mployed to
exclude problematic regions with putatively high content
of non-phylogenetic signal. The ‘‘less stringent selection’’ cri-
teria, as deﬁned in the program, were applied. The align-
ments are available at http://www.deep-phylogeny.org/ or
from the authors upon request.
2.7. Phylogenetic analyses
The appropriate model of protein evolution was deter-
mined using ProtTest (Abascal et al., 2005). The content
Missing data in the non-edited concatenated alignment and the alignment edited with GBlocks
Non-edited total % Edited with GBlocks total %
Cnidaria Hydra magnapapillata 1265 25.2 965 22.7
Podocoryne carnea 307 6.1 249 5.8
Echinodermata Strongylocentrotus purpuratus 208 4.1 154 3.6
Chordata Ciona intestinalis 6 0.1 0 0
Homo sapiens 000 0
Mus musculus 000 0
Rattus norwegicus 000 0
Xenopus tropicalis 000 0
Nematoda Ascaris suum 32 0.6 10 0.2
Haemonchus contortus 312 6.2 258 6.1
Meloidogyne paranaensis 1100 21.9 926 21.7
Tardigrada Hypsibius dujardini 929 18.5 749 17.6
Onychophora Epiperipatus sp. 1317 26.2 1002 23.5
Arthropoda Acanthoscurria gomesiana 1265 25.2 1039 24.4
Aedes aegypti 000 0
Anopheles gambiae 000 0
Apis mellifera 7 0.1 7 0.1
Boophilus microplus 637 12.7 616 14.5
Callinectes sapidus 2564 51.1 2186 51.3
Daphnia magna 337 6.7 226 5.3
Bombyx mori 000 0
Drosophila melanogaster 000 0
Penaeus monodon 167 3.3 81 1.9
Rhipicephalus appendiculatus 2366 47.1 1939 45.5
Tribolium castaneum 44 0.9 18 0.4
Annelida Lumbricus rubellus 91 1.8 6 0.1
Mollusca Crassostrea sp. 666 13.3 485 11.4
Mytilus galloprovincialis 1105 22.0 935 22.0
Argopecten irradians 489 9.7 383 9.0
Relative proportions refer to total alignment lengths of 4259 (non-edited) and 5021 (edited with GBlocks) positions, respectively.
944 F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951
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of phylogenetic information of the alignments was esti-
mated by the likelihood mapping approach as implemented
in Tree-Puzzle 5.0 (Strimmer and von Haeseler, 1997).
Maximum likelihood trees were reconstructed using
IQPNNI (Vinh and von Haeseler, 2004). Bootstrap-repli-
cates were generated with Seqboot from the PHYLIP pack-
age. Bayesian phylogenetic analyses were performed by
MrBayes 3.1.2 (Huelsenbeck and Ronquist, 2001). Two
parallel runs were performed. Metropolis-coupled Markov
chain Monte Carlo (MCMCMC) sampling was carried
out with one cold and three heated chains that were run
for 3.1 million generations with random starting trees.
Every 100th tree was sampled. To infer whether the
MCMC chains had reached equilibrium, we analyzed the
results with TRACER (http://evolve.zoo.ox.ac.uk/software.
html?id=tracer). The ﬁrst 1000 sampled trees (100,000
generations) were discarded to allow for burn-in of the
Markov chain. From the remaining trees, the majority rule
consensus tree was computed. Increasing the burn-in time
to 10,000 trees (1 million generations) had no eﬀect on tree
topologies and posterior probabilities of the splits.
3.1. ESTs from the velvet worm Epiperipatus
A cDNA phage library was constructed from mRNA
of two velvet worm (Epiperipatus sp.) specimens (Kusche
et al., 2002). We obtained 1873 single pass reads of the 5
ends of randomly selected clones. Clustering resulted in
833 contigs (239 contigs were represented by more than
one EST, 594 were singletons). Epiperipatus contigs that
matched mitochondrial sequences in a TBlastX search
were then excluded from further analys es (nine contigs).
The remaining 824 contigs were compared with the
non-redundant (nr) protein database at NCBI employing
a BlastX search. A total of 428 of the contigs (51.9%)
matched to known proteins with E-value of less or equal
, 345 contigs (41.9%) had hits at a cut-oﬀ at
(41.4%) and 261 contigs returned hits with E
lower than 10
The top hits of the contigs at the nr database were
analyzed for taxonomic distribution. Approximately the
same proportion of ESTs had high-score with proteins
from Chordata (40.9–43.3%) and Arthropoda (38.3–
40.3%). Only a minor proporti on of the ESTs had their
best BlastX hits with sequences from other phyla such as
Mollusca, Nematoda or Annelida ( Fig. 1). Employing
the classiﬁcation by GOﬁgure (http://udge-
nome.ags.udel.edu/frm_go.html), the contigs were
assigned to functional categories according to the BlastX
results (Fig. 2). The proportion of the categor ies showed
a broad spectrum of gene functions. Most of the best
hits matched to enzymes (27–33%), transporters, recep-
tors and other binding proteins (18–19%) or structural
proteins (13–16%). Eight to 14% of the best hit proteins
had no known function.
3.2. Assembling the ribosomal proteins of Metazoa
The BlastX searches with the Epiperi patus contigs (cut-
oﬀ: E <10
) revealed similarities to 27 ribosomal pro-
teins. No additional hits to ribosomal sequences were
obtained when we relaxed the stringency of our search.
The human and Drosophila orthologs of these proteins
were obtained from the Unigene database. Using Blast
searches, we further retrieved the sequences of these ribo-
somal proteins from the genome assemblies of mouse
(Mus musculus), rat (Rattus norvegicus), malaria mosquito
(Anopheles gambiae) and honeybee (Apis mellifera). Then
the EST databases of other metazoan taxa were searched
for the selected ribosomal proteins by TBlastN. We
obtained positive hits for a large number of species and
eventually selected 22 taxa on the basis of sequence quality,
taxonomic distribution and sequence coverage (Table 1).
The ESTs of each species and each protein were individu-
ally assembled into contigs. For further analyses, the
Fig. 1. Taxonomic classiﬁcation of the best BlastX hits. Percentage of
sequences with top hits in various phyla with three diﬀerent E-value cut-
oﬀs: (a) 10
; (b) 10
; (c) 10
F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951 945
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ribosomal protein sequences of the closely related bivalve
species Crassostrea gigas and C. virginica were combined
(i.e. Crassostrea sp.; Supplemental Table 1). Thus the ﬁnal
alignments included 29 taxa.
3.3. The phylogeny of Metazoa based on ribosomal proteins
The amino acid sequences for the 27 ribosomal pro-
teins were aligned with ClustalX and the resulting align-
ments were adjusted by hand. Likelihood mapping, a
method that visualizes the phylogenetic signal within a
sequence alignment (Strimmer and von Haeseler, 1997),
resulted in a high proportion of unresolved quartets
(3.8–65.9%; Supplemental Fig. 1; Supplemental Table 2).
Correspondingly, phylogenetic analyses of the alignments
of individual proteins using maximum parsimony or
Bayesian methods did not result in any meaningful trees
(data not shown). Therefore, a concatenated ‘‘supergene’’
alignment of the 27 ribosomal proteins was constructed
that covers 5021 amino acids. In few taxa, some
ribosomal proteins or parts of them were not represented
in the databases. These positions were coded as ‘‘missing
data’’ (a total of 10.4% of the data were missing in this
alignment); additional 7.7% of the data were indels.
After excluding noisy regions with Gblocks (Cas tresana,
2000), the edited alignment covers 4259 amino acid
positions with 9.9% missing data and 0.3% indels (for
details cf. Supplemental Table 1). Likelihood mapping
analyses showed that in both the 5021 and the 4259
amino acids alignments >99.2% of the quartets were
fully resol ved and none of the trees was star-like
(Supplemental Fig. 2).
We employed maximum likelihood and Bayesian analy-
ses to construct phylogenet ic trees from the 5021 amino
acid (Fig. 3) and the 4259 amino acid alignments (Fig. 4).
The rtRev model (Dimmic et al., 2002) with a discrete
gamma model for varying substitution rates among sites
assuming four rate categories ( Yang, 1994) was found the
best model of amino acid evolution. The MCMC chains
of Bayesian inference were run for 3.1 million generations.
The two Cnidaria (Hydra, Podocoryne) were considered as
outgroup that roots the Bilateria. In all trees Lophotrocho-
zoa, represented here by Mollusca (Crassostrea, Argopecten
and Mytilus) and an annelid worm (Lumbricus), and
Ecdysozoa (Epiperipatus, Nematoda, Tardigrada,
Fig. 2. Gene ontology of best BlastX hits. Sequences were classiﬁed
according to GOﬁgure. Three diﬀerent E-value cut-oﬀs were applied: (a)
; (b) 10
; (c) 10
Fig. 3. Bayesian inference of Metazoan phylogeny based on the 5021
amino acid alignment of ribosomal proteins. The rtRev model of amino
acid evolution was assumed as prior setting. Posterior probabilities (ﬁrst
number) and bootstrap support values of maximum likelihood estimates
(second number) are depicted at the nodes.
946 F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951
Author's personal copy
Arthropoda) were found monophyletic. While Chordata
were monophyletic, Deuterostomia were not. In some
trees, Chordata were found closer to Protostomia than to
Echinodermata (Strongylocentrotus). This result may be
attributed to insuﬃcient taxon sampling or to the usage
of a too distant outgroup. Within the Ecdysozoa, the
topologies of the trees wer e essentially the same, with the
exception of Epiperipatus. The monophyly of insects and
crustaceans (Pancrustacea or Mandibulata, as Myriapoda
were included in the analysis), received high bootstrap
and Bayesian support, as well as the grouping of the bran -
chiopod crustacean (Daphnia) with the insects. Nematodes
(Ascaris, Haemonchus, and Meloidogyne) and the tardi -
grade Hypsibius form ed together a clade in all analyses.
In all analys es, Epiperipatus was included in the Ecdysozoa,
although its exact position in the trees depended on the
alignment and the applied method. Employing the concat-
enated 5021 amino acid align ment, Epiperipatus was found
positioned either as sistergroup to the Euarthropoda, but
neither in ML nor in Bayesian analyses the support was
signiﬁcant (Fig. 3). After removing low structure regions
with GBlocks (4259 amino acid alignment), ML and Bayes-
ian analyses showed the same topology (Fig. 4), i.e, with
Epiperipatus included in the Euarthropoda and being in
sistergroup position to the Chelicerata.
4.1. Analysing onychophoran gene expression
Although Onychophora are a phylogenetically impor-
tant metazoan phylum, their representation in sequence
databases is still poor. We have constructed and analysed
the ﬁrst EST database from an onychophoran species.
About half of the contigs had hits with known proteins.
The onychophoran ESTs covers a broad range of func-
tional categories (Fig. 2), as anticipated from a cDNA
library prepared from entire animals. Interestingly, we
observed the same number of best hits with Arthropoda
and Chordata (Fig. 1). The high number of arthropod hits
was expected because of the relationship of Onychophora
and Arthropoda. The similarly high number of hits from
Chordata may be due to the bias of sequences per phylum
within the databases. Of course such a distribution is only
of limited phylogenetic information, since it does not take
diﬀerent evolution rates into account. Thus, it will be
biased towards an overrepresentation of more slowly
evolving species. It is nevertheless interesting to note that
only 1% of the Epiperipatus ESTs had their best hits with
an annelid protein. Together with the molecular phyloge-
netic analyses (Figs. 3 and 4) this ﬁnding further argues
against Onychophora as a ‘‘bridge’’ between Arthropoda
and Annelida (Ax, 2000) and weakens the support for the
Articulata hypothesis (Snodgrass, 1938).
4.2. The utility of ESTs to analyze metazoan phylogeny
It is still a controversial issue to what extent reliability of
tree reconstruction beneﬁts from maximizing either the
number of taxa or the number of characters studied (cf.
Hillis, 1998; Rosenberg and Kumar, 2003; Philippe et al.,
2004). Most phylogenetic studies that investigate a large
number of taxa include alignments of single genes or a lim-
ited number of selected genes (e.g., Giribet et al., 2001;
Regier and Shultz, 2001; Mallatt et al., 2004). Recent stud-
ies have revealed that increasing the number of characters
has a more positive eﬀect on tree credibility than the inclu-
sion of additional taxa (Mitchell et al., 2000; Rosenberg
and Kumar, 2003 Rokas and Carroll, 2005). These analy-
ses emphasize the beneﬁt of independent characters, i.e.
the individual sequences of diﬀerent genes.
ESTs are a cost-eﬀective mean to maximize the amount of
sequence data (Rudd, 2003). At the ﬁrst glance, phyloge-
nomic approaches that employ EST data appear to be lim-
ited because the sampled taxa may not contain an
overlapping set of data. One approach to overcome this
problem is the creation of ‘‘chimeric’’ sequences, i.e.tocom-
bine genes from diﬀerent species to analyse phylogeny at a
higher taxonomic level (e.g., Philippe et al., 2005; Baurain
et al., 2007). Here we focussed on the analysis of only ribo-
somal proteins, which are well-represented in the EST data-
sets from all species (see below). As we have shown, even a
mid-size EST approach results in suﬃcient sequence
Fig. 4. Bayesian inference of Metazoan phylogeny based on the 4259
amino acid alignment of ribosomal proteins. The rtRev model of amino
acid evolution was assumed as prior setting. Posterior probabilities (ﬁrst
number) and bootstrap support values of maximum likelihood estimates
(second number) are depicted at the nodes.
F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951 947
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information that allows a largely reliable reconstruction of
metazoan systematics at the level of phyla. This agrees with
previous approaches, which have sh own that ESTs may be
used for the placement of Xenoturbella within Deuterosto-
mia (Bourlat et al., 2006) or for the resolution of the relation-
ships among eukaryote crown-groups (Philippe et al., 2004).
4.3. Metazoan phylogeny based on ribosomal proteins
Ribosomal proteins seem particularly suitable for phylog-
eny reconstruction by means of an EST-based approach for
mainly two reasons. First, translation of mRNA into protein
is a crucial process in the cells’ metabolism, which takes place
at the ribosomes. Therefore, it is expected that ribosomal
proteins are among the most abundant proteins in a typical
cell. In fact , as many as 27 out of the 833 contigs (3.2 %)
obtained from our EST dataset code for ribosomal proteins.
In addition to actin and tubulin, ribosomal proteins are the
only cDNA sequences that can consistently be found in the
EST datasets from various species. Second, as noted by Phi-
lippe et al. (2004), there are essentially no paralogs of these
genes within Metazoa, making them prime candidates inves-
tigating ancient relationships among Metazoa. Hansmann
and Martin (2000) studied prokaryote phylogeny on the
basis of genome-derived sequences of ribosomal proteins,
Philippe et al. (2004) used the EST-derived sequences of ribo-
somal proteins (and other data) to investigate the relation-
ships among Eukaryota, and Hughes et al. (2006) applied a
similar phylogenomic approach within the beetles
The systematics of Metazoa as presented here was derived
from the analyses of 27 ribosomal proteins, which are repre-
sented in the Epiperipatus ESTs. The phylogeny of the Met-
azoa could not be inferred on the basis of single ribosomal
proteins, but only afte r construction of a concatenated align-
ment. This agrees with similar observations in other studies,
which found that the use of single or few genes is insuﬃcient
for the conﬁdent resolution of many clades of the Metazoa
(e.g., Bapteste et al., 2002; Rokas et al., 2005). In initial anal-
yses, maximum parsimony (MP) or neighbour-joinin g (NJ)
trees did not resolve the relationships among Metazoa with
suﬃcient conﬁdence (not shown). This result appears to be
in line with recent observation that even a large dataset (50
genes) investigated by MP or maximum likelihood (ML)
methods was not suitable to resolve metazoan relationships
(Rokas et al., 2005). This was interpreted as ‘‘a signature of
closely spaced series of cladogenetic events’’. However, oth-
ers do not agree with this notion (Baurain et al., 2007) and in
fact we found that at least Bayesian phylogenetic inference
led to reasonable trees with only minor controversial branch-
ing patterns (Figs. 3 and 4).
4.4. The position of the Onychophora and support for the
The Ecdysozoa hypothesis states that Arthropoda and
Onychophora are related to other moulting phyla such as
Tardigrada, Nematoda, Nematomorpha, Priapulida,
Loricifera and Kinorhyncha (Aguinaldo et al., 1997;
Adoutte et al., 2000; Ruiz-Trillo et al., 2002; Giribet,
2003; Mallatt et al., 2004). This hypothesis also suggests
that the Annelida, previously allied with Arthropoda (i.e.
‘‘Articulata’’), are included in a supertaxon referred to as
‘‘Lophotrochozoa’’, which includes Sipunculida, Mol-
lusca, Tentaculata, some Platyhelminthes along with
other phyla (Adoutte et al., 2000). Our trees based on
a concatenated alignment of ribosomal proteins add fur-
ther support to this concept. Arthropoda were found
associated with Onychophora, Tardigrada and Nematoda
and this clade received high support in ML and Bayesian
analysis (Figs. 3 and 4). Accordingly, the monophyly of
the Lophotrochozoa (i.e. Mollusca and Annelida) is well
The position of the Onychop hora within the Ecdysozoa
remains unclear. Only the Bayesian analysis of the full
alignment placed Epiperipatus, as expected, as sistergroup
to the Euarthropoda. ML and Bayesian trees of the
reduced dataset (4259 amino acid alignment) joined Epipe-
ripatus with the Chelicerates. Recent neuroanatomical
studies have in fact suggested a relationship of Onychopho-
ra and Chelicerata (Strausfeld et al., 2006). How ever, this
topology contradicts all other morphological (Nielsen,
2001; Brusca and Brusca, 2003) and molecular studies (Bal-
lard et al., 1992; Boore et al., 1995; Edgecombe et al., 2000;
Giribet et al., 2001; Kusche et al., 2002). The inclusion of
the available 50 other clearly orthologous ribosomal pro-
teins in the alignment (29 taxa, 77 proteins; 14,487 amino
acid positions and 11,656 positions after treatment with
GBlocks) resulted in trees with the same overall branching
pattern within the Metazoa, but did not increase the reli-
ability of the position of Epiperipatus (data not shown).
Additional taxon sampling, which sh ould also include
other arthropods such as Myriapoda and the Peripatopsi-
dae, will probably improve conﬁdence.
4.5. Are tardigrades Cycloneuralia?
Tardigrades, here represented by Hypsibius, are enig-
matic, tiny animals that harbour morphological characters
which are reminiscent of both Arthropoda and Cyclone-
uralia (i.e. Nematoda, Nematomorpha, Priapulida, Loricif-
era and Kinorhyncha) (Nelson, 2002; Brusca and Brusca,
2003; Giribet, 2003). Arthropod-like characters of Tardi-
grada include the segmented body, limbs and ladder-like
central nervous syst em (Westheide and Rieger, 1996; Brus-
ca and Brusca, 2003; Giribet, 2003). On the other hand, the
structures of mouth, pharynx, cuticle and sensory organs of
tardigrades rather resemble those of Cycloneuralia (Giri-
In the ﬁrst molecular phylogenetic analyses based on
18S rRNA, tardigrades were found associated with
Arthropoda (Garey et al., 1996; Giribet et al., 1996). Garey
et al. (1996) placed the nematodes at the base of the meta-
zoan tree, while in the analysis of Giribet et al. (1996) this
948 F. Roeding et al. / Molecular Phylogenetics and Evolution 45 (2007) 942–951
Author's personal copy
taxon was not included. Aguinaldo et al. (1997) found tar-
digrades nested within the now paraphyletic Arthropoda.
In a later study of 133 18S rRNA sequences, the tardi-
grades formed a sistergroup of Nematoda, although the
support for that clade remained uncertain (Giribet and
Ribera, 2000). Most recently, based on combined 18S
and 28S rRNA data, Mallatt and Giribet (2006) associated
Tardigrada with Onychophora.
In our analyses, both ML and Bayesian methods of the
complete and edited versions of the ribosomal protein
alignment, provide additional support for the view that
within the monophyletic Ecdysozoa, but suggest that Tar-
digrada (Hypsibius) are more closely related to Nematoda
than to Arthropoda (Figs. 3 and 4). This result also agrees
with recent trees obtained by Philippe et al. (2005) and
Baurain et al. (2007). If conﬁrmed, this placement suggests
that the arthropod-like characters of Tardigrada, such as
the segmented body, limbs and ladder-like central nervous
system may be plesiomorphic characters for Ecdysozoa.
However, additional evidence is required to conﬁrm the
phylogenetic aﬃnities of water bears.
Our analyses show that even a mid-sized EST dataset
contains suﬃcient phylogenetic information for a useful
and largely reliable reconstruction of the phylogeny of
Metazoa (see also e.g., Philippe and Telford, 2006). As
one example, we were able to support the Ecdysozoa and
Lophotrochozoa as valid taxa with high conﬁdence. In par-
ticular, ribosomal protein sequences, which are abundant
in EST data, and which include suﬃcient phylogenetic
information to resolve metazoan relationships, appear to
be promising (cf. Philippe et al., 2004, 2005; Bourlat
et al., 2006).
However, the present EST approach embraces several
problems. First, we could not place some taxa with suﬃ-
cient conﬁdence. We already mentioned Epiperipatus (Ony-
chophora), but similar problems emerged with Schistosoma
mansoni (Platyhelminthes) (not shown). In some cases (par-
ticularly Platyhelminthes), sequences tend to experience
very fast evolution rates, which has led to the erosion of
phylogenetic information. Second, further improvement
of tree reconstruction can be expected from the develop-
ment of better models of sequence evolution. This is partic-
ularly true for the ribosomal proteins, for which to date no
speciﬁc model exists. In our analyses, the rtRev model,
which had actually been derived from retroviral and other
reverse transcriptase-containing sequences (Dimmic et al.,
2002), was predicted to have the highest log likelihood
score. Third, due to the lack of ESTs in the database s,
many impor tant invertebrate phyla were missing in our
analyses. It might be expected that additional ESTs from
many taxa will be obtained within the next few years. These
data will not only allow the placement of missing taxa, but
will also increase the reliability of the tree (cf. Baurain
et al., 2007).
This work has been supported by the Deutsche Fors-
chungsgemeinschaft within the Priority Project ‘‘Deep
Metazoan Phylogeny’’ (Bu 956/8-1 and Ha1628/8-1).
Arndt von Haeseler and Ingo Ebersberger are also sup-
ported by the Vienna Science and Technology Fund
(WWTF). We thank Julia Markl (Heidelberg) and Rob-
pﬂin (Hamburg) for their help with data
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