Phylogenetic Position of Nemertea Derived from Phylogenomic Data
Torsten H. Struck and Frauke Fisse
FB05 Biology/Chemistry, AG Zoology, University of Osnabrück, Osnabrück, Germany
Nemertea and Platyhelminthes have traditionally been grouped together because they possess a so-called acoelomate
organization, but lateral vessels and rhynchocoel of nemerteans have been regarded as coelomic cavities. Additionally,
both taxa show spiral cleavage patterns prompting the placement of Nemertea as sister to coelomate Protostomia, that is,
either to Neotrochozoa (Mollusca and Annelida) or to Teloblastica (Neotrochozoa plus Arthropoda). Some workers
maintain a sister group relationship of Nemertea and Platyhelminthes as Parenchymia because of an assumed homology
of Götte’s and Müller’s larvae of polyclad Platyhelminthes and the pilidium larvae of heteronemerteans. So far,
molecular data were only able to significantly reject a sister group relationship to Teloblastica.
Although phylogenomic data are available for Platyhelminthes, Annelida, Mollusca, and Arthropoda, they are
lacking for Nemertea. Herein, we present the first analysis specifically addressing nemertean phylogenetic position using
phylogenomic data. More specifically, we collected expressed sequence tag data from Lineus viridis (O.F. Müller, 1774)
and combined it with available data to produce a data set of 9,377 amino acid positions from 60 ribosomal proteins.
Maximum likelihood analyses and Bayesian inferences place Nemertea in a clade together with Annelida and Mollusca.
Furthermore, hypothesis testing significantly rejected a sister group relationship to either Platyhelminthes or Teloblastica.
The Coelomata hypothesis, which groups coelomate taxa together to the exclusion of acoelomate and pseudocoelomate
taxa, is not congruent with our results. Thus, the supposed acoelomate organization evolved independently in Nemertea
and Platyhelminthes. In Nemertea, evolution of acoely is most likely due to a secondary reduction of the coelom as it is
found in certain species of Mollusca and Annelida. Though looking very similar, the Götte’s and Müller’s larvae of
polyclad Platyhelminthes are not homologous to the pilidium larvae of heteronemerteans. Finally, the convergent
evolution of segmentation in Annelida and Arthropoda is further substantiated.
Introduction
Nemertea or ribbon worms are primarily benthic marine invertebrates, but a few of the approximately 900 described species inhabit pelagic, freshwater, or terrestrial
habitats and some are symbionts of other marine invertebrates (Brusca RC and Brusca GJ 2003). These unsegmented worms range in length from less than 1 cm to
several meters and their bodies are quite flexible so that
some species can increase their contracted body length several times (Brusca RC and Brusca GJ 2003). Specimens of
Lineus longissimus (Gunnerus, 1770) are among the longest
known invertebrates with a length of over 30 m (Turbeville
1996). Due to their general dorsoventrally flattened, and only
moderately cephalized, habitus, the first described nemerteans were regarded as turbellarian flatworms. Only in
the 19th century was the taxon Nemertea erected and placed
outside Platyhelminthes (Schultze 1851; Minot 1876; Coe
1943; Hyman 1951; Brusca RC and Brusca GJ 1990). The
monophyly of Nemertea is well established and primarily
based on their unique eversible proboscis, which is surrounded by a rhynchocoel (e.g., Turbeville 1996; Brusca
RC and Brusca GJ 2003; Jenner 2004b).
Based on traditional concepts of secondary body cavities, bilaterian taxa have been categorized into 3 levels of
organization: acoelomate, pseudocoelomate, and coelomate
(Hyman 1951; Jenner 2004b). Both, Nemertea and Platyhelminthes, were regarded as acoelomate and accordingly
grouped together (Hyman 1951). Additionally, the acoelomate and pseudocoelomate organization were regarded as
ancestral and the coelomate one as the supposed synapomorphy of Coelomata (Hennig 1979; Blair et al. 2002;
Key words: Nemertea, phylogenomics, Lophotrochozoa, Eutrochozoa, Parenchymia, Teloblastica.
E-mail: [email protected].
Mol. Biol. Evol. 25(4):728–736. 2008
doi:10.1093/molbev/msn019
Advance Access publication January 24, 2008
Ó The Author 2008. Published by Oxford University Press on behalf of
the Society for Molecular Biology and Evolution. All rights reserved.
For permissions, please e-mail: [email protected]
Philip et al. 2005). However, the lateral vessels of the closed
circulatory system and the rhynchocoel of nemerteans have
been proven to represent coelomic cavities, which arise
by schizocoely (Turbeville and Ruppert 1985; Turbeville
1986). Furthermore, Nemertea and Platyhelminthes show
spiral cleavage patterns and thus most authors place them
in a clade, which also contains other taxa exhibiting spiral
cleavage such as Annelida, Mollusca, and Entoprocta
(Brusca RC and Brusca GJ 1990, 2003; Meglitsch and
Schram 1991; Ax 1995; Nielsen 1995, 2001; Rouse and
Fauchald 1995; Haszprunar 1996; Nielsen et al. 1996;
Cavalier-Smith 1998; Garey and Schmidt-Rhaesa 1998;
Sundberg et al. 1998; Zrzavy et al. 1998, 2001; Giribet
et al. 2000; Sorensen et al. 2000; Peterson and Eernisse
2001; Zrzavy 2003; Jenner 2004b).
The taxon composition of this clade, as well as the specific position of Nemertea within it, is controversial. Discussions based on morphological data center on 3 major
topics, which result in 3 distinct hypothesis concerning sister group relationships of Nemertea: Are the Götte’s and
Müller’s larvae of polyclad Platyhelminthes and the pilidium
larvae of heteronemerteans homologous (e.g., Nielsen
1995)? Are the coelomic cavities of Nemertea homologous
with coelomic spaces of other taxa, for example, Annelida
(e.g., Turbeville 1986)? Finally, though not directly related
to Nemertea, is Arthropoda part of this clade or have they to be
placed within Ecdysozoa (e.g., Brusca RC and Brusca GJ
1990; Peterson and Eernisse 2001)?
Mainly based on similarities of larval features such as
a reduced hyposphere, the lack of an anus and the shape of
the larval ciliary bands a close relationship of Nemertea and
Platyhelminthes as Parenchymia is still maintained by some
authors (Nielsen 1995, 2001; Nielsen et al. 1996; Sorensen
et al. 2000). Additional characters supporting Parenchymia
are the mode of development of the adult nervous system
and the lack of chitin and chitinase (Nielsen 1995, 2001).
These authors interpret coelomic cavities of Nemertea as
their autapomorphy.
Phylogenetic Position of Nemertea 729
Several authors proposed a sister group relationship of
Nemertea to Neotrochozoa, which comprise Mollusca,
Annelida, Sipuncula, Echiura, Siboglinidae (also known
as Pogonophora and Vestimentifera), and Myzostomida
(Zrzavy et al. 1998; Giribet et al. 2000; Peterson and
Eernisse 2001; Zrzavy 2003; Jenner 2004b). Increasing
evidence of both morphological and molecular data shows
that the latter 4 taxa are annelid subtaxa and thus as a consequence Neotrochozoa refers to the sister group relationship of Annelida and Mollusca (e.g., Bartolomaeus 1995;
McHugh 1997; Hessling 2002; Eeckhaut et al. 2003;
Wanninger et al. 2005; Bleidorn et al. 2007; Struck et al.
2007). Among others, the placement of Nemertea as sister
to Neotrochozoa is substantiated by the development of the
lateral coelom due to schizocoely from mesoderm germ
bands derived from the 4d mesoteloblast (e.g., Turbeville
1986, 2002; Eernisse et al. 1992; Peterson and Eernisse
2001; Jenner 2004b). Additionally, in Neotrochozoa and
Nemertea, the 3a and 3b blastomeres give rise to the ectomesoderm, whereas in polyclad Platyhelminthes, the ectomesoderm is derived from the 2b cell (Turbeville 2002).
For a detailed discussion of several other synapomorphic
characters, see the critical reviews of Jenner (2004b) and
Turbeville (2002). This clade comprising Neotrochozoa
and Nemertea was further substantiated by combined analyses of 18S and morphological data (e.g., Giribet et al.
2000; Peterson and Eernisse 2001) and christened
Eutrochozoa (Peterson and Eernisse 2001). Haszprunar
(1996) also included Entoprocta in this clade.
Other authors have suggested that Nemertea are sister
to Teloblastica (Brusca RC and Brusca GJ 1990; Meglitsch
and Schram 1991; Ax 1995; Rouse and Fauchald 1995;
Sundberg et al. 1998). Teloblastica comprise, at a minimum,
neotrochozoan taxa plus Arthropoda, which were previously seen as the sister to Annelida. Therefore, preference
by workers for either Neotrochozoa or Teloblastica as sister
to Nemertea rather depends how they view the relative
positions of Annelida and Arthropoda; those supporting
Lophotrochozoa/Ecdysozoa (e.g., Halanych 2004) prefer
Neotrochozoa, whereas Articulata hypothesis (e.g., Brusca
RC and Brusca GJ 2003) is consistent with the Teloblastica
hypothesis. (Note Entoprocta [Ax 1995] or Tardigarda
[Meglitsch and Schram 1991] have been included into
Teloblastica as well.) To ensure consistency of names of
taxa assemblages throughout the manuscript, we generally
followed Jenner (2004b) even if the cited authors themselves did not use this specific name for a certain taxa assemblage. Furthermore, Platyhelminthes is used herein in
the sense of Halanych (2004) excluding Acoelomorpha.
Morphological and molecular evidence support polyphyly
of traditional Platyhelminthes with Acoelomorpha being
basal bilaterians and the remaining Platyhelminthes being
part of Lophotrochozoa (see Halanych 2004).
Molecular data so far strongly support only a placement of Nemertea within Lophotrochozoa and thus reject
a sister group relationship to Teloblastica (Turbeville et al.
1992; Erber et al. 1998; Balavoine et al. 2002; Mallatt and
Winchell 2002; Ruiz-Trillo et al. 2002; Anderson et al.
2004; Halanych 2004; Peterson and Butterfield 2005;
Passamaneck and Halanych 2006; Turbeville and Smith
2007; Helmkampf et al. 2008). Depending on the respective
analyses and the genes used Nemertea are closely related to
different taxa within this clade, for example, Platyzoa
(Passamaneck and Halanych 2006) or Mollusca (Erber
et al. 1998; Zrzavy et al. 1998; Turbeville and Smith
2007; Helmkampf et al. 2008). However, none of these relationships are substantially supported.
Recent phylogenomic approaches have generally improved the robustness of molecular phylogenetic reconstructions (Philippe et al. 2005; Philippe and Telford
2006; Baurain et al. 2007; Hausdorf et al. 2007), but adequate genomic data are still lacking for Nemertea. Herein,
we present analyses using expressed sequence tag (EST)
data of the nemertean Lineus viridis (O.F. Müller, 1774)
to address specifically these major outstanding issues of
the origin of Nemertea. The results based on 9,377 amino
acids from 60 ribosomal proteins place Nemertea within
Eutrochozoa as sister to Mollusca. Hypotheses testing significantly rejected the Parenchymia hypothesis as well as
a sister group relationship to Teloblastica.
Materials and Methods
Isolation of RNA and Library Construction
The nemertean L. viridis was collected at the marine
biological station Wadden Sea Station Sylt in List (North
Sea Island Sylt, Germany) and frozen using liquid nitrogen.
Total RNA was extracted employing TRIzol (Invitrogen,
Karlsruhe, Germany). Quality of total RNA was visually
checked on agarose gel, and mRNA was subsequently captured using Dynabead (Invitrogen). The cDNA library was
constructed at the Max Planck Institute for Molecular Genetics in Berlin by primer extension, size fractioning, and
directional cloning applying Invitrogen’s CloneMiner technology, using the vector pDONR222. A total of 4,545
clones containing cDNA inserts were sequenced from the
5# end on the automated capillary sequencer systems ABI
3730 XL (Applied Biosystems, Darmstadt, Germany) and
MegaBace 4500 (GE Healthcare, Hunohen, Germany) using
BigDye chemistry (Applied Biosystems).
EST Processing
EST processing was accomplished at the Center for
Integrative Bioinformatics in Vienna. Sequencing chromatograms were first base called and evaluated using the Phred
application (Ewing et al. 1998). Vector, adaptor, poly-A,
and bacterial sequences were removed employing the
software tools Lucy (www.tigr.org), SeqClean (http://
compbio.dfci.harvard.edu/tgi/software), and CrossMatch
(http://www.phrap.org), respectively. Repetitive elements
were subsequently masked with RepeatMasker. Clustering
and assembly of the clipped sequences were performed using the TIGCL program package (http://compbio.dfci.
harvard.edu/tgi/software) by first performing pairwise comparisons (MGIBlast) and a subsequent clustering step
(CAP3). Low-quality regions were then removed by Lucy.
Finally, contigs were tentatively annotated by aligning
them pairwise with the 25 best hits retrieved from National
Center for Biotechnology Information’s nonredundant
730 Struck and Fisse
protein database using the BlastX algorithm (http://www.
ncbi.nlm.nih.gov). Alignment and computation of the resulting match scores on which annotation was based were
conducted by GeneWise (Birney et al. 2004) in order to account for frameshift errors. The EST data used in our analyses have been deposited in GenBank under the accession
numbers EU302527–EU302586.
Sequence Analyses and Ribosomal Proteins Alignment
Ribosomal protein sequences were extracted from the
nemertean EST data using the human ribosomal proteome
(retrieved from the Ribosomal Protein Gene Database;
http://ribosome.med.miyazaki-u.ac.jp) as search template
during local Blast searches (TBlastN algorithm and an
e value , e10 as match criterion). Sixty out of the 79 ribosomal proteins were found in the EST data of L. viridis.
Observed sequences were checked for assembly errors
by visual inspection and by comparison with corresponding
sequences of related taxa and translated into amino acid sequences. Additional ribosomal protein data were retrieved
from the alignments compiled by Hausdorf et al. (2007).
Amino acids were used for the phylogenetic analyses instead of nucleotides for several reasons. First, the greater
number of character states in amino acid data (21 compared
with 4 for nucleotides) minimizes the potential of convergence. Second, protein data lack synonymous substitutions,
which are a major source of homoplasy in nucleotide data.
Third, amino acid alignments are generally less variable
than nucleotide alignments and therefore more suitable
for reconstruction of deep nodes.
All ribosomal protein sequences obtained were
aligned by the ClustalW algorithm using default parameters
(Thompson et al. 1994). The resulting 60 ribosomal protein
alignments were inspected and only adjusted manually for
obviously misaligned positions using GeneDoc (Nicholas
KB and Nicholas HB 1997). Questionably aligned positions were eliminated with GBlocks (Castresana 2000). Default parameters were used with allowed gap positions set to
‘‘with half’’ and conversation of the flanking positions set to
70%. The alignment of the concatenated sequences was deposited at http://www.treebase.org (accession number: S1979).
Phylogenetic Analysis
Baurain et al. (2007) showed that Platyhelminthes,
Nematoda, and Tardigarda introduce long branch problems,
which may mislead the placement of these taxa even in
phylogenomic analyses. Therefore, 2 data sets were analyzed. In one data set, nematodes and tardigrades were excluded to circumvent possible artificial misplacements of
Platyhelminthes or Platyzoa. For both data sets and all
7 implemented criteria, ProtTest (Abascal et al. 2005) determined rtREV þ C þ F as the most appropriate substitution
model. Maximum likelihood (ML) analyses were conducted with Treefinder (Jobb et al. 2004; Jobb 2007). Confidence values for the edges of the ML tree were computed
by applying expected likelihood weights (ELWs) (Strimmer
and Rambaut 2002) to all local rearrangements of tree
topology around an edge (LR-ELW; 1,000 replications).
Table 1
Topology Test Results of Nemertean Relationships Using the
Expected Likelihood Weight (ELW) Test
Nemertea, sister to
Mollusca
Annelida
Neotrochozoa (Annelida þ Mollusca)
Neotrochozoa þ Entoprocta
Platyhelminthes
Platyzoa (Platyhelminthes þ Syndermata)
Teloblastica (Neotrochozoa þ Arthropoda)
Teloblastica þ Tardigarda
Teloblastica þ Entoprocta
32 taxa
30 taxa
0.5262
0.4258
0.0140*
0.0064*
0.0205*
0.0070*
,0.0001*
,0.0001*
,0.0001*
0.3919
0.4835
0.0275*
0.0904
0.0062*
0.0002*
,0.0001*
n.a.
,0.0001*
NOTE.—Asterisks indicate values for the topologies not included in the 0.95
confidence set (i.e., ELWs of the trees with the highest confidence levels that add up
to 0.95). n.a. 5 not applicable.
To test a priori phylogenetic hypotheses (table 1), we
constrained trees and used the ‘‘resolve multifurcations’’
option of Treefinder to obtain the ML tree for a specified
hypothesis. Next, we investigated whether the ML trees
for these hypotheses are part of the confidence set of
trees applying the ELWs method with 50,000 replications
(Strimmer and Rambaut 2002).
Bayesian inferences (BIs) based on the site heterogeneous CAT model (Lartillot and Philippe 2004) were performed using PhyloBayes v2.1c (Blanquart and Lartillot
2006). For each data set, 2 independent chains were run simultaneously for 5,000 points each. Chain equilibrium was
estimated by plotting the log likelihood and the alpha parameter as a function of the generation number. The first
1,000 points were consequently discarded as burn-in for
both data sets. According to the divergence of bipartition
frequencies, both chains of each data set reached convergence (maximal difference 0.528 or 0.447, respectively;
mean difference 0.012 or 0.011). For each data set, taking
every 10th sampled tree a 50% majority rule consensus tree
was finally computed using both chains.
Results
Alignment of the concatenated sequences of 60 ribosomal proteins included 9,377 amino acid positions. Depending on the method (ML or BI), as well as on the
data set (32 taxa or 30 taxa), the topologies of the best trees
are different mainly with respect to the position of Platyhelminthes and Syndermata. Therefore, the results of all analyses are shown (figs. 1 and 2).
All phylogenetic analyses show Nemertea as a eutrochozoan taxon either as sister to monophyletic Mollusca
(figs. 1 and 2A) or to monophyletic Annelida sensu Struck
et al. (2007) (fig. 2B). Thus, placement of Nemertea within
Eutrochozoa is independent from inclusion of the longbranched tardigrades and nematodes in the analyses. However, nodal support is only strong by posterior probabilities
(PP: 0.98, fig. 1A; PP: 0.96, fig. 2A). Eutrochozoa are either
sister to monophyletic Bryozoa sensu lato (figs. 1B and 2B)
or part of a basal polytomy comprising also Platyhelminthes
and bryozoan taxa (figs. 1A and 2A). In both BI’s, Platyhelminthes are part of a basal polytomy in the lophotrochozoan
Phylogenetic Position of Nemertea 731
FIG. 1.—Nemertea are a eutrochozoan taxon in the analyses with 32 taxa. Phylogenetic analyses were performed on the basis of 9,377 amino acid
positions derived from 60 concatenated ribosomal proteins. Nemertea are highlighted. Dashed lines indicate paraphyletic taxa in that tree. (A) BI.
Posterior probabilities are shown at the nodes. Syn. 5 Syndermata. (B) ML. Approximate bootstrap support values (LR-ELW) are shown at the nodes.
clade and Syndermata render Ecdysozoa paraphyletic (figs.
1A and 2A). Both ML analyses recover Platyzoa but in the
analysis based on 32 taxa Platyzoa are within Ecdysozoa
(fig. 1B) and in the one based on 30 taxa sister to Lophotrochozoa (fig. 2B).
For both data sets, hypothesis testing did not differentiate between a sister group relationship of Nemertea to
either Mollusca or Annelida but nemerteans as sister to
Neotrochozoa was rejected (table 1). A sister group relationship to Neotrochozoa plus Entoprocta was clearly rejected with the data set comprising 32 taxa. The
Parenchymia hypothesis as well as a sister group relationship to Teloblastica with or without either Tardigrada or
Entoprocta were significantly rejected.
Discussion
Nemerteans as a Eutrochozoan Taxon
Nemertea is a eutrochozoan taxon closely related to
Annelida and Mollusca. These results further substantiate
previous assertions that the coelomic cavities of Nemertea,
Annelida, and Mollusca are of common ancestry and thus
homologous (e.g., Turbeville and Ruppert 1985; Turbeville
1986; Eernisse et al. 1992; Peterson and Eernisse 2001;
Jenner 2004b). The lateral vessels of the nemertean circulatory system are surrounded by a continuous lining of mesoderm cells, which are connected to each other by
adhaerens and septate junctions and possess, if at all, only
rudimentary cilia (Turbeville and Ruppert 1985; Turbeville
1986). In these ultrastructural details, the vessels are similar
to peritoneal linings of coelomic cavities, for example, of
Annelida (Turbeville 1986; Bartolomaeus 1994; Rieger and
Purschke 2005). Furthermore, development of nemertean
lateral vessels and coelom formation in annelids both occur
by schizocoely (Potswald 1981; Turbeville 1986). The vessels ‘‘begin as solid epithelial bands, which secondarily cavitate, resulting in a cell-lined channel’’ (Turbeville 1986).
Finally, the mesodermal bandlets of the eutrochozoan taxa
derive from the 4d mesoteloblast and give rise to lateral coelom cavities by schizocoely (Henry and Martindale 1998;
Peterson and Eernisse 2001; Jenner 2004b).
The homology of coelomic cavities of Nemertea, Annelida, and Mollusca has been doubted because in the latter
2 taxa, the mesodermal bands also give rise to body musculature (Bartolomaeus 1994). However, at least parts of
the circulatory system of an interstitial nemertean Cephalothrix sp. are similar to a myoepithelium forming portions
of the body-wall musculature (Turbeville 2002). Additionally, in 2 other nemerteans, parts of the mesothelium are not
732 Struck and Fisse
FIG. 2.—Nemertea are a eutrochozoan taxon in the analyses with 30 taxa. Nematoda and Tardigarda were excluded from the analyses to
circumvent possible artificial misplacements of Platyhelminthes or Platyzoa. Phylogenetic analyses were performed on the basis of 9,377 amino acid
positions derived from 60 concatenated ribosomal proteins. Nemertea are highlighted. Dashed lines indicate paraphyletic taxa in that tree. (A) BI.
Posterior probabilities are shown at the nodes. Syn. 5 Syndermata. (B) ML. Approximate bootstrap support values (LR-ELW) are shown at the nodes.
separated from adjacent muscle cells by an intervening extracellular matrix, which is similar in organization to some
annelids (Turbeville 2002). Nielsen (1995, 2001) has argued that the lack of associated metanephridia (or otherwise
open nephridial connections) as well as the nonsegmental
organization suggest that nemertean circulatory system is
not homologous to neotrochozoan coelomic cavities. However, the secondary circulatory system of Hirudinea
(Annelida), which is derived from coelomic cavities, is
of nonsegmental nature and the ciliated funnels of their
metanephridia, if present, are not connected to the nephridial ducts. The latter prevents that blood cells are continuously lost to the environment. Overall, the circulatory
system of Nemertea is comparable but not homologous
to the secondary circulatory system of Hirudinea.
Thus, the traditional delineation of body organization
into acoelomate, pseudocoelomate, and coelomate is rather
a classification scheme for a morphological character than
the basis for phylogenetic clades (Halanych and Passamaneck
2001; Jenner 2004b). Additionally, the significant
rejection of Parenchymia indicates that similar body organization evolved independently in Platyhelminthes and
Nemertea in contrast to traditional concepts. The phylogenetic position of Nemertea recovered here is consistent with
the morphological hypothesis (Turbeville 1986) that
their body cavity has been secondarily reduced to the point
of assuming an acoelomate-like state. Secondary reductions
of the coelom can also be seen, for example, in Hirudinea,
several other annelids, Mollusca, and Arthropoda (e.g.,
Rieger and Purschke 2005). Therefore, Nemertea could also
be regarded as coelomate. However, it may be more informative to drop these categories and instead focus more on
identifying homologous elements of coelomic cavities. Differing developmental origins of coelomic cavities in the different bilaterian lineages cast doubts on the homology of
the coelom across bilaterians (Minelli 1995; Salvini-Plawen
and Bartolomaeus 1995; Nielsen 2001). Therefore, the possession of a coelom is a weak character to unite all coelompossessing taxa as Coelomata (Hennig 1979; Blair et al.
2002; Philip et al. 2005). Furthermore, our results are consistent with numerous previous molecular studies that suggest Coelomata, as traditionally recognized, is not a real
clade (e.g., Giribet et al. 2000; Peterson and Eernisse
2001; Halanych 2004; Philippe et al. 2005; Passamaneck
and Halanych 2006; Baurain et al. 2007; Hausdorf et al.
2007).
In hoplonemerteans and palaeonemerteans juveniles,
nonspecialized uniformly ciliated planktonic larva develops
directly into adult stages (e.g., Norenburg and Stricker
2002; Maslakova et al. 2004b). This mode of development
Phylogenetic Position of Nemertea 733
with a so-called planuliform larva is thought to belong to
the ground pattern of Nemertea (Thollesson and Norenburg
2003; Maslakova et al. 2004b). In contrast, all other eutrochozoan taxa plus Entoprocta possess a trochophore larva,
which is defined by the presence of a prototroch derived
from trochoblasts (Rouse 1999). Maslakova et al.
(2004a; 2004b) showed that the ‘‘planuliform’’ larva of
Carinoma tremaphoros Thompson 1900 (Palaeonemertea)
possesses a preoral belt of large ciliated cells, which are cell
cleavage arrested and are derived from 4 primary trochoblast. Thus, the development of the belt is similar to the
one of prototrochs in Annelida and Mollusca (Maslakova
et al. 2004b). Therefore, though uniformly ciliated the planuliform larva is likely to be homologous to a trochophore
larva (Maslakova et al. 2004b). Our results further warrant
this conclusion.
A sister group relationship of Nemertea to monophyletic Neotrochozoa is not very likely contrary to previous
combined analyses (Giribet et al. 2000; Peterson and
Eernisse 2001). Nemertea are placed as sister to either
Annelida or Mollusca, though weakly supported. Neither
position is well supported by morphological data, but
Cavalier-Smith (1998) united Annelida and Nemertea as
Vermizoa due to the possession of closed blood vessels,
ciliated larvae without bivalved shells, and 2 ventrolateral
or one primitively paired ventral nerve cord. Unfortunately,
these features are wide spread within Bilateria and circulatory systems are not homologous across bilaterians. As
mentioned above, Nemertea use a reduced coelom derivative for blood transport, whereas Annelida (except for
Gnathobdelliformes and Pharyngobdelliformes, Hirudinea)
use spaces in the extracellular matrix between 2 adjoining
epithelia, a morphology typical of invertebrates in general
(Westheide 1997; Hartenstein and Mandal 2006). Comparatively, a close relationship of Nemertea to Mollusca has
been found in several studies using different molecular
markers (intermediate filament: Erber et al. 1998; 18S:
Zrzavy et al. 1998; mtDNA: Turbeville and Smith 2007;
and 7 nuclear genes: Helmkampf et al. 2008). However,
as in our analyses support for such an association is weak
(PP: 0.98, fig. 1A; LR-ELW: 55, fig. 1B; and PP: 0.96, fig.
2A). Because posterior probabilities are generally higher
than bootstrap values approximated herein by the LRELW method (see figs. 1 and 2 and Huelsenbeck et al.
2002) and are less reliable measurements of support than
bootstrap values (e.g., Suzuki et al. 2002; Lewis et al.
2005), the term ‘‘significant support’’ should refer only
to bootstrap support above 95 or to results based on hypothesis tests such as the conducted ELW test with defined
P values. Therefore, the position of Nemertea within Eutrochozoa remains uncertain.
To address the position of Nemertea future studies
could combine this data set of ribosomal proteins with data
of both additional genes and taxa. Though phylogenomic
approaches have been proposed to end incongruence
(e.g., Gee 2003; Rokas et al. 2003), recent studies showed
that as for single or few gene analyses increased taxon sampling is also necessary to increase robustness and minimize
systematic errors due to, for example, long branches (e.g.,
Hillis et al. 2003; Soltis et al. 2004; Philippe and Telford
2006; Baurain et al. 2007). Thus, data of additional
nemerteans, mollusks, annelids, and other lophotrochozoan
taxa such as Brachiopoda and Phoronida, which are not
covered yet, might improve the phylogenetic reconstructions. This could also increase the robustness and nodal
support of the present topologies (figs. 1 and 2) in general,
which is still low at several deep nodes. Especially, the position of long-branched taxa such as Nematoda, Tardigarda,
and Platyhelminthes (Baurain et al. 2007) can be possibly
determined more accurately.
Nemertea and Platyhelminthes/Platyzoa
A sister group relationship of Nemertea and Platyhelminthes can be clearly rejected. Because some studies propose monophyletic Platyzoa, which also comprises
Platyhelminthes (Cavalier-Smith 1998; Garey and
Schmidt-Rhaesa 1998; Giribet et al. 2000; Passamaneck
and Halanych 2006), we also tested if Nemertea were sister
to Platyzoa. This can also be clearly rejected (table 1).
Therefore, the homology of the polyclad Götte’s and
Müller’s larvae and the heteronemertean pilidium larvae
(Nielsen 1995, 2001; Nielsen et al. 1996; Sorensen et al.
2000) is rejected by the present data. This homology proposal depends on the assumption that both larval types belong to the ground pattern of Nemertea as well as
Platyhelminthes (Turbeville 2002; Jenner 2004b). However, most turbellarian groups are direct developers without
larvae. Besides polyclads, larvae are only present in Catenulida and parasitic forms, which are very different from the
Götte’s and Müller’s larvae (e.g., Nielsen 1995). In nemerteans, only Heteronemertea and the palaeonemertean Hubrechtella develop indirectly with a pelagic pilidium
larva (e.g., Nielsen 1995). However, due to the possession
of this larva Hubrechtella is likely to be a heteronemertean
(Cantell 1969; Norenburg 1985, 1988). A recent molecular
study supported a sister group relationship of Heteronemertea and Hubrechtella as well as a highly derived position of
this clade within Nemertea (Thollesson and Norenburg
2003). Assuming that the pilidium larva belongs to the nemertean ground pattern would mean the larva has been secondarily reduced 3 times. Therefore, the pilidium larvae are
an autapomorphy of this clade and not a feature of the nemertean ground pattern (Turbeville 2002; Thollesson and
Norenburg 2003; Jenner 2004b; Maslakova 2004b). The
same can be shown for the Götte’s and Müller’s larvae
and polyclad Platyhelminthes (Zrzavy et al. 1998; Giribet
et al. 2000; Turbeville 2002; Jenner 2004b). Finally,
Turbeville (2002) showed that the addition of just 2 morphological characters (i.e., gliointerstitial cell system and
schizocoelous coelom) to the data set of Nielsen (2001) rendered Parenchymia paraphyletic and placed Nemertea as
sister to Teloblastica. In the analyses of Nielsen (2001), Parenchymia was supported by possessing a reduced larval
hyposphere.
The other 2 characters supporting Parenchymia, adults
with only an apical nervous system and the lack of chitin
and chitinase (Nielsen 1995), are contentious. Adults with
only an apical nervous system can also be found in some
mollusks (see Jenner 2004b). As for lack of chitin or chitinase, the interpretation of absent morphological characters
734 Struck and Fisse
in a phylogenetic context as either plesiomorphic absent or
secondary lost and thus apomorphic absent is difficult (e.g.,
Purschke et al. 2000; Collin and Cipriani 2003; Jenner
2004a; Struck 2006).
Nemertea and Teloblastica
Not surprisingly, a sister group relationship of Nemertea to Teloblastica could be significantly rejected because
Arthropoda is not closely related to neotrochozoan taxa
(e.g., Halanych 2004; Philippe et al. 2005; Baurain et al.
2007; Hausdorf et al. 2007; Helmkampf et al. 2008).
Additionally, Hausdorf et al. (2007) could significantly reject the Articulata hypothesis (e.g., Nielsen 1995; Brusca
RC and Brusca GJ 2003). Furthermore, none of our analyses constraining Teloblastica with or without Entoprocta
or Tardigarda recovered Articulata. Arthropoda (even including Tardigarda) were always placed as the most basal
taxon within the constrained Teloblastica. Thus, segmentation evolved independently in annelids and arthropods
(Seaver and Kaneshige 2006). This is further substantiated
by the derived position of Annelida within Lophotrochozoa
in our analyses. Furthermore, unsegmented worms such as
Sipuncula and Echiura are placed within Annelida showing
the variability of the character complex segmentation
(McHugh 1997; Peterson and Eernisse 2001; Bleidorn
et al. 2003, 2006; Struck et al. 2007).
Acknowledgments
We would like to thank J. von Döhren and T.
Bartolomaeus (Free University Berlin) for providing tissue
material. We are also grateful to M. Kube and R. Reinhardt
(Max Planck Institute for Molecular Genetics, Berlin) for
the construction and sequencing of cDNA libraries and
to I. Ebersberger, S. Strauss, and A. von Haeseler (Max
F. Perutz Laboratories, Center for Integrative Bioinformatics, Vienna) for the processing of our ESTs. B.
Hausdorf (University of Hamburg), T. Hankeln, and B.
Lieb (both University of Mainz) provided data of ribosomal
proteins prior to public release. A. Müller and A. Paululat
(both University of Osnabrück) provided access to a Linux
server or a MacPro to run PhyloBayes v2.1c, respectively.
We thank K. M. Halanych (Auburn University), B.
Hausdorf (University of Hamburg), and G. Purschke
(University of Osnabrück) for comments on first drafts
of the manuscript. This study was funded by the priority
program ‘‘Deep Metazoan Phylogeny’’ of the Deutsche
Forschungsgemeinschaft (grant STR 683/3-1 to Torsten
H. Struck).
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Billie Swalla, Associate Editor
Accepted January 20, 2008