Illuminating the Base of the Annelid Tree Using
Transcriptomics
Anne Weigert,*,1 Conrad Helm,1 Matthias Meyer,2 Birgit Nickel,2 Detlev Arendt,3 Bernhard Hausdorf,4
Scott R. Santos,5 Kenneth M. Halanych,5 Günter Purschke,6 Christoph Bleidorn,y,1 and
Torsten H. Strucky,7
1
Institute of Biology, Molecular Evolution and Animal Systematics, University of Leipzig, Leipzig, Germany
Department of Evolutionary Genetics, Max Planck Institute for Evolutionary Anthropology, Leipzig, Germany
3
EMBL Heidelberg, Heidelberg, Germany
4
Zoological Museum, University of Hamburg, Hamburg, Germany
5
Department of Biological Sciences, Molette Biology Laboratory for Environmental and Climate Change Studies, Auburn University
6
Department of Zoology and Developmental Biology, University of Osnabrueck, Osnabrueck, Germany
7
Zoological Research Museum Alexander Koenig, Bonn, Germany
y
These authors contributed equally to this work.
*Corresponding author: E-mail: [email protected].
Associate editor: Gregory Wray
2
Abstract
Annelida is one of three animal groups possessing segmentation and is central in considerations about the evolution of
different character traits. It has even been proposed that the bilaterian ancestor resembled an annelid. However, a robust
phylogeny of Annelida, especially with respect to the basal relationships, has been lacking. Our study based on transcriptomic data comprising 68,750–170,497 amino acid sites from 305 to 622 proteins resolves annelid relationships,
including Chaetopteridae, Amphinomidae, Sipuncula, Oweniidae, and Magelonidae in the basal part of the tree.
Myzostomida, which have been indicated to belong to the basal radiation as well, are now found deeply nested
within Annelida as sister group to Errantia in most analyses. On the basis of our reconstruction of a robust annelid
phylogeny, we show that the basal branching taxa include a huge variety of life styles such as tube dwelling and deposit
feeding, endobenthic and burrowing, tubicolous and filter feeding, and errant and carnivorous forms. Ancestral character
state reconstruction suggests that the ancestral annelid possessed a pair of either sensory or grooved palps, bicellular
eyes, biramous parapodia bearing simple chaeta, and lacked nuchal organs. Because the oldest fossil of Annelida is
reported for Sipuncula (520 Ma), we infer that the early diversification of annelids took place at least in the Lower
Cambrian.
Introduction
Annelids, or segmented worms, comprise over 21,000 recognized species found in marine, freshwater, and terrestrial habitats. The group has been central in debates on major
transitions in animal evolution such as the development of
segmentation, evolution of the nervous system, transitions to
a terrestrial lifestyle, and origins and diversifications of larval
types (Purschke 1999; Rouse 1999; Seaver 2003; Jekely et al.
2008). To address such topics, a well-supported phylogeny
of Annelida is needed to assess ancestral conditions and
to allow discrimination between alternative hypotheses.
Unfortunately, relationships among basal annelid lineages
are still poorly understood (Struck et al. 2011; Kvist and
Siddall 2013), hindering our understanding of early annelid
evolution. Because they usually lack hard body parts, the fossil
record of the early annelid radiation is sparse and provides
little resolution on the early annelid radiation. Although some
trace and tube fossils from the Ediacara fauna (635–542 Ma)
reportedly resemble polychaetes (Glaessner 1976a, 1976b;
Retallack 2007), the oldest accepted annelid fossils are reported from the Maotianshan Shale, Sirius Passet, and the
Burgess Shale from the Lower Cambrian about 520 Ma
(Huang et al. 2004; Morris and Peel 2008; Vinther et al. 2011).
Several hypotheses have been proposed regarding morphological characteristics of the last common ancestor of
Annelida, but currently there is little consensus on this
issue. According to Westheide (1997), there are two major
types of competing scenarios.
The first type of scenario describes evolution of annelids
from simple to complex, where the stem species lack head
appendages and parapodia have simple chaetae as an adaptation to burrowing (Clark 1969; Westheide 1997). This view
was inspired by the traditional division of Annelida into
Polychaeta and Clitellata (oligochaetes and leeches) and received support by morphological-cladistic analyses (Rouse
and Fauchald 1997). In accordance with this hypothesis, the
ß The Author 2014. 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]
Mol. Biol. Evol. 31(6):1391–1401 doi:10.1093/molbev/msu080 Advance Access publication February 23, 2014
1391
Article
Key words: Annelida, Annelid fossils, Cambrian, next generation sequencing, phylogenomics.
MBE
Weigert et al. . doi:10.1093/molbev/msu080
former “Archiannelida,” which were considered a primitive
group due to the lack of parapodia and chaetae in several
taxa, have been debated as very close to the annelid stem
species (Hermans 1969). However, molecular and morphological data show that “Archiannelida” constitute a polyphyletic assemblage of independently evolved putatively
progenetic lineages, which converged on a simplified body
plan (Fauchald 1977; Purschke and Jouin 1988).
The second type of scenario describes the opposite direction of evolution from a more complex errant, epibenthic
ancestor with well-developed head appendages, parapodia,
and chaetae toward more simple forms by modifications,
reductions, and losses (Storch 1968). Investigating morphological or molecular data, which found Clitellata, Echiura, and
Siboglinidae nested within Polychaeta and Myzostomida as
well as Sipuncula within the annelid radiation, in part substantiated this view (McHugh 1997; Struck et al. 2008;
Bleidorn et al. 2009; Zrzavy et al. 2009; Helm et al. 2012).
These analyses indicated that reductions of characters frequently occurred in Annelida (Purschke et al. 2000; Bleidorn
2007).
At present, phylogenetic hypotheses lack consensus as to
which annelid taxa are to be placed at the base of the tree
(McHugh 2000; Bleidorn et al. 2003; Rousset et al. 2007; Struck
et al. 2008; Zrzavy et al. 2009). Additionally, some analyses
struggled to recover a monophyletic Annelida and lacked
resolution for deeper nodes perhaps due to using limited
numbers of loci. Only recently Struck et al. (2011) presented
a robust backbone of the annelid tree based on a phylogenomic analysis including expressed sequence tag (EST) libraries of several annelid families. In this analysis, ancestral
character state reconstructions suggested that the last
common ancestor possessed only one pair of head appendages (i.e., grooved palps), nuchal organs, and parapodia with
simple and internalized supporting chaetae. From this ancestor, evolution progressed to both more complexly and more
simply organized annelids. In that study, the basal part of
the annelid tree comprises only three taxa: Sipuncula,
Myzostomida, and Chaetopteridae. Although congruent
with some previous studies (Struck et al. 2008; Zrzavy et al.
2009; Dordel et al. 2010), support for relationships among
these basal branching taxa depended on the reconstruction
method used (Struck et al. 2011; Kvist and Siddall 2013).
Moreover, crucial taxa with respect to basal relationships of
Annelida were lacking, for example, Oweniidae and
Magelonidae (Rousset et al. 2007; Zrzavy et al. 2009).
To illuminate the basal radiation of Annelida and to test
their phylogenetic relationships, we extended the taxon sampling by generating transcriptome libraries for 22 additional
annelid taxa, focusing on potential basal branching taxa. In
total, our analyses now comprise 60 annelid species, representing 39 annelid families. This is about one-third of the total
number of approximately 125 annelid families (Fauchald and
Rouse 1997; Jamieson and Ferraguti 2006; Siddall et al. 2006),
covering 6 out of 35 clitellate families and 33 out of 80 polychaete families. Furthermore, we expanded the data matrix
to 170,497 amino acid positions derived from 622 genes. So
far, this is the largest data set available for annelids based on
1392
next-generation sequencing data to provide a robust annelid
phylogeny.
Results
We generated seven data subsets ranging from 68,750 to
170,497 amino acid positions derived from 305 to 622
genes (table 1). Gene coverage of the matrix increased from
0.378 to 0.547 for the 79-taxa data subsets, from 0.375 to 0.549
in the 77-taxa data subsets, and from 0.402 to 0.555 for the
72-taxa data subset (table 1). Topologies obtained from all
seven data subsets are nearly identical except for the positions
of Myzostomida and Orbiniidae (fig. 1, supplementary figs.
S1–S6, Supplementary Material online). Errantia and
Sedentaria are recovered in all analyses with Clitellata
deeply nesting within Sedentaria. The relationships in the
basal part of the annelid tree are robust regardless of data
set employed and supported by moderate (bootstrapping
[BS] 70) to significantly high (BS 95) bootstrap values,
especially in the analyses without Myzostomida (fig. 1, supplementary figs. S4 and S5, Supplementary Material online).
Sipuncula together with Amphinomidae are sister group to
Pleistoannelida. Chaetopteridae is sister to this clade.
Magelonidae and Oweniidae form a monophyletic group
and together are the sister group of all other annelids.
Monophyly of Annelida is recovered with significantly high
(BS 99) support values in all analyses without Myzostomida
(fig. 1, supplementary figs. S4 and S5, Supplementary Material
online). By including Myzostomida, monophyletic Annelida
are supported by moderate (BS 70, supplementary fig. S2,
Supplementary Material online) to significantly high (BS 99,
supplementary fig. S1, Supplementary Material online) support values in the analyses comprising the highest number of
genes (table 1: 79-taxa data subsets 1.5 and 1). In these analyses, Myzostomida fall within Pleistoannelida as sister taxon of
Errantia, which comprises Phyllodocida and Eunicida (supplementary figs. S1 and S2, Supplementary Material online).
However, in the analysis of the 79-taxa data subset 2
(table 1) comprising 305 genes and 68,903 aa positions,
long-branched Myzostomida are closely related to outgroup
taxa Cycliophora and Entoprocta (supplementary fig. S3,
Supplementary Material online). The problem of analyzing
the phylogenetic position of the long branching
Myzostomida has been shown before (Bleidorn et al. 2007)
and is also reflected by their LB scores in our present analyses.
In all three 79-taxa data subsets, the two myzostomid species
as well as the cycliophoran Symbion and the two entoproct
species of Pedicellina are among the six taxa with highest LB
scores. Leaf stability analyses also reflected the problematic
placement of myzostomids (supplementary table S6,
Supplementary Material online). Because of the possible
long branch attraction of Myzostomida and outgroups, we
created a seventh data set comprising 72 taxa where we excluded all outgroups except Mollusca, Nemertea, and
Brachiopoda. Because the analysis of the 79-taxa data
subset 2 was the only one resulting in paraphyletic
Annelida, with Myzostomida nesting within outgroups (supplementary fig. S3, Supplementary Material online), we applied the same weighting parameter to the 72-taxa data set as
MBE
Base of the Annelid Tree . doi:10.1093/molbev/msu080
Table 1. Data Sets Generated with MARE and Analyzed in this Study
with Additional Information on Number of Genes, Amino Acid
Positions, Gene Coverage, and information Content for Each Data
Subset.
Data Set
Unreduced
Subset 1
Subset 1.5
Subset 2
No. of
Taxa
79
79
79
79
No. of
Genes
2,339
620
419
305
aa
Positions
751.842
169.332
104.410
68.903
Gene
Coverage
0.378
0.489
0.520
0.547
Information
Content
0.090
0.193
0.227
0.255
Unreduced
Subset 1
Subset 1.5
Subset 2
77
77
77
77
2,339
622
421
308
746.984
170.497
103.420
68.750
0.375
0.487
0.519
0.549
0.090
0.193
0.227
0.256
Unreduced
Subset 2
72
72
2,339
305
756.207
69.157
0.402
0.555
0.096
0.261
NOTE.—On the two unreduced data sets comprising either 77 or 79 taxa, three
different weighing parameters (MARE, = 1, 1.5, or 2) were applied resulting in six
data subsets. On the unreduced data set comprising 72 taxa, only one weighing
parameter (MARE, = 2) was applied. Overall seven data subsets were generated.
to the data set with 79 taxa (MARE = 2) using the same
genes (supplementary table S5, Supplementary Material
online) to investigate the influence of LBA on the position
of Myzostomida (72-taxa data subset 2). As a result, we find
monophyletic Annelida with significantly high (BS 95) support and Myzostomida nest within Errantia as sister taxon of
Eunicida (supplementary fig. S6, Supplementary Material
online). Within Sedentaria, relationships among taxa stay
robust across all seven data sets except for the position of
Orbiniidae. The leaf stability indices of the basal branching
annelids were above 0.96 for all data sets, and, hence, the
lower support values observed in the analyses of the 79taxa data subset 1.5 (supplementary fig. S2, Supplementary
Material online) can be attributed to the unstable position of
Myzostomida as discussed earlier.
We also tested whether alternative positions for
the basal branching taxa (i.e., Sipuncula, Amphinomidae,
Chaetopteridae, Oweniidae, and Magelonidae) could be rejected. The tested options were placement of Sipuncula outside Annelida, inclusion of Amphinomida within Errantia, and
Chaetopteridae, Oweniidae, and Magelonidae as members of
Sedentaria (supplementary table S4, Supplementary Material
online). All nine tested a priori hypotheses, which are mainly
based on the traditional systematic view and on morphology
could be significantly rejected (supplementary table S4,
Supplementary Material online).
Moreover, we explored if paralogy or xenology, compositional heterogeneity, lack of data, or long branches influenced
the placement of these taxa. The plot of per-site log likelihoods (psL) of each constrained analysis in comparison to
the best tree showed that the phylogenetic signal for each
topology (the constraint and the best tree) is scattered across
the whole alignment and not concentrated in certain genes
(data dryad: files S1–S9). Hence, we find no evidence that the
support for the position of Oweniidae, Magelonidae,
Chaetopteridae, Amphinomidae, and Sipuncula in the basal
part of the annelid tree is due to paralogous or xenologous
sequences. Wilcoxon–Mann–Whitney tests also revealed no
significant differences in the distributions of both RCFV values
and proportion of missing sequence data for the “basal” annelid taxa and all other taxa in the tree in all seven data
subsets (supplementary table S3, Supplementary Material
online). Thus, the basal position of these annelid taxa is
most likely not due to shared compositional heterogeneity
or similar degrees of missing data.
Because we extended the taxon sampling and certain ancestral character traits of the annelid stem species were not
present in all basal branching taxa, like the nuchal organs and
grooved palps, we repeated the ancestral state reconstruction
of Struck et al. (2011) including our new taxa and results. The
results of the new reconstructions are generally identical to
the ones of Struck et al. (2011) in, among others, the presence
of a pair of peristomial palps, bicellular eyes, and biramous
parapodia bearing simple chaetae (supplementary table S7,
Supplementary Material online, fig. 3). However, three major
differences could be observed: 1) it is uncertain if the pair of
peristomial palps is of the solid or the grooved kind; 2) internalized supporting chaetae are lacking; and 3) nuchal organs
are absent. The other differences were either uncertain characters becoming a certain state or vice versa.
Discussion
Annelid Relationships
Phylogenomic analyses provided robust support for basal
annelid relationships including Oweniidae, Magelonidae,
Chaetopteridae, Sipuncula, and Amphinomidae (fig. 2).
Interestingly, Magelonidae and Oweniidae represent the
sister group to all other annelids with strong support. Our
results are surprisingly congruent with a hypothesis made by
Rieger (1988), where he suggested that members
of Oweniidae closely resemble the annelid stem species and
should be placed near the base of the annelid tree. His view
was inspired by the presence of certain morphological
characters in Oweniidae, which are absent in other annelid
taxa and considered as plesiomorphic rather than as derived:
the presence of a largely intraepidermal nervous system
(Bubko and Minichev 1972), the mitraria larva possessing
monociliated epidermal cells and nephridia with
deuterostome similarities (Smart and Von Dassow 2009),
and presence of monociliated epidermal cells in adults
(Gardiner 1978; Westheide 1997). Because monociliated epidermal cells were thought to be absent in Annelida, they can
elsewhere be found in members of the lophotrochozoan
groups Gnathostomulida, Gastrotricha, Phoronida, and
Brachiopoda (Rieger 1976; Gardiner 1978; Nielsen 2002). For
Oweniidae, they could be confirmed in larvae and adults of
Owenia fusiformis (Gardiner 1978) and in adults of the genus
Myriowenia (Westheide 1997). Interestingly, larvae of
Magelona mirabilis (Magelonidae) likewise possess an epidermis made up of monociliated cells (Bartolomaeus 1995). A
close relationship of Magelonidae and Oweniidae was also
recently proposed by Capa et al. (2012) based on a cladistic
analyses of morphological characters. However, care must be
1393
Weigert et al. . doi:10.1093/molbev/msu080
MBE
FIG. 1. Best maximum likelihood (ML) tree of the RAxML analysis using the LG + I + G model of the data set comprising 77 taxa and including 421
genes with 104,410 amino acid positions (MARE settings = 1.5). Only bootstrap values above 50 are shown.
1394
Base of the Annelid Tree . doi:10.1093/molbev/msu080
MBE
FIG. 2. Overview and phylogenetic placement of the five basal branching lineages in the annelid tree. (A) Cladogram of the phylogenetic relationships
within Annelida without myzostomids based on all seven analyses (with Orbiniidae as sister group to Siboglinidae and Cirratuliformia as in six of
the seven analyses). (B) Sipuncula: Phascolosoma scolops from Sydney, Australia. (C) Amphinomidae: Eurythoe complanta var. mexicana from
the Sea of Cortez, Mexico. Photo: Carlos Sanchez Ortiz (Universidad Autónoma de Baja California Sur). (D) Chaetopteridae: Chaetopterus
variopedatus from Roscoff, France. (E) Oweniidae: Owenia fusiformis from Saint-Efflam, France. (F) Magelonidae: Magelona johnstoni from SaintEfflam, France.
taken in evaluating characters as apomorphic or plesiomorphic. For instance, an intra- or basiepidermal nervous
system is widespread in annelids, and homoplasy cannot be
ruled out (Bullock and Horridge 1965; Purschke 2002; Orrhage
and Müller 2005). Moreover, retention of a plesiomorphic
character does of course in no way support the phylogenetic
position of its bearer. Given our results and the scattered
appearance of monociliated epidermis cells in Metazoa
(Rieger 1976), the former could well be a synapomorphy for
the clade of Magelonidae and Oweniidae within Annelida.
Moreover, we find important changes regarding the phylogenetic position of certain taxa with respect to the analyses
of Struck et al. (2011), highlighting the importance of extended taxon sampling and extended gene coverage to recover a robust phylogeny beyond the basal relationships. By
increasing the number of analyzed genes in our analyses,
Myzostomida fell within Annelida as part of Errantia and
are not part of the basal radiation as in Struck et al. (2011).
We find that bootstrap values for monophyly of Annelida and
a myzostomid-Errantia sister group relationship increase with
1395
Weigert et al. . doi:10.1093/molbev/msu080
the number of analyzed genes. The phylogenetic position of
myzostomids was always problematic due to long-branch
attraction with outgroup taxa, but several previous analyses
using multiple markers and rare genomic changes strongly
support Myzostomida as part of the annelid radiation as well
(Bleidorn et al. 2007; Hartmann et al. 2012; Helm et al. 2012).
In contrast to Struck et al. (2011), Orbiniidae and
Amphinomidae are placed differently. By including more
species of each taxon, the grouping of Aphroditiformia
(Phyllodocida), Orbiniidae, and Amphinomidae was not recovered: Instead, Orbiniidae grouped within Sedentaria,
whereas Amphinomidae branched off in the basal part of
the tree as sister to Sipuncula. These changes also resulted
in the monophyly of Phyllodocida, which was paraphyletic in
Struck et al. (2011). The previous positions of Aphroditiformia
(Phyllodocida), Orbiniidae, and Amphinomidae were most
likely due to low gene coverage and a shared paralog by
these three (Struck 2013). Notably, the basal branching position of Amphinomidae had been found in a few previous
molecular studies but received little attention so far
(Hausdorf et al. 2007; Struck et al. 2008). Also, an early branching of Chaetopteridae, which has been indicated previously
(Struck et al. 2007; Struck et al. 2008; Bleidorn et al. 2009;
Zrzavy et al. 2009; Dordel et al. 2010), was confirmed with
high support. Placement of Sipuncula within Annelida is
strongly supported by our analyses and substantiates previous
studies (Bleidorn et al. 2009; Mwinyi et al. 2009; Dordel et al.
2010). Because of the absence of segmentation, parapodia,
and chaetae, as well as typical head and body appendages,
the phylogenetic position of Sipuncula was under discussion
since their description (Schulze et al. 2005). Sperling et al.
(2009) recently suggested the absence of segmentation in
Sipuncula likely to be primary rather than secondary based
on microRNAs and the fossil record. Because the amount of
characters used in their analysis was low and the taxon sampling extremely poor (three annelid species), with crucial
basal branching taxa lacking, placement of Sipuncula as
sister group to the three other annelids was not surprising.
On the contrary, our analyses, strongly supporting Sipuncula
as part of the annelid radiation, suggest that absence of segmentation may be a derived character state, which was indicated in a previous study by immunohistological
investigations of the nervous system (Kristof et al. 2008).
Character Evolution in Annelids
The controversy of the direction of annelid evolution is crucially linked to character states assumed for their last common
ancestor (Clark 1969; Westheide 1997). In this discussion, the
vast morphological diversity and different life modes of annelid taxa have to be taken into account as, for example, the
basal branching taxa include tube-dwelling, deposit-feeding
Oweniidae, endobenthic, burrowing Magelonidae, tubicolous,
filter-feeding Chaetopteridae, unsegmented, burrowing
Sipuncula, and errant, carnivorous Amphinomidae.
According to Struck (2011), the ancestral annelid was an
intermediate form between a simple- or complex-bodied
ancestor and represented a “microphagous surface
1396
MBE
deposit-feeder crawling upon and through soft-bottom habitats.” Although generally in agreement with these conclusions, results of the reconstructions herein showed slight
differences to the ones of Struck et al. (2011) in the uncertainty of the kind of peristomial palps as either solid or
grooved, lack of internalized supporting chaetae, and absence
of nuchal organs. Thus, in comparison to these previous studies (Struck 2011; Struck et al. 2011), the ancestral annelid was
slightly more simply organized.
Palps, regardless of whether they emerge from the prostomium or peristomium, are innervated similarly and, therefore,
regarded to be homologous (Fauchald and Rouse 1997;
Orrhage and Müller 2005). They can be divided into grooved
ciliated feeding palps and ventral, tapering sensory palps; the
former bifunctional (sensory and feeding) and the latter
purely sensory. Within the basal radiation, sensory palps are
found in Amphinomidae only. In Magelonidae, a derived type
of grooved palps may be present, which develops in ontogeny
after shedding of the primary ones (Wilson 1982). Therefore,
the palps of the ancestral annelid most likely were of the
grooved (ciliated) type having a dual function of gathering
food and sensing (Struck 2011), although this hypothesis
could not be resolved by ancestral reconstruction.
The lack of supporting chaetae is much more in line with
the known fossil record of the Cambrian as such chaetae were
not observed in these fossils (Vinther et al. 2011; EibyeJacobsen and Vinther 2012).
Although almost all annelid taxa possess nuchal organs,
their lack in the annelid ground pattern has been proposed
before, based on the traditional position of Clitellata as sister
to Polychaeta (Rouse and Fauchald 1997; Purschke 2002).
However, as Clitellata are deeply nested within polychaetes
(Rousset et al. 2007; Zrzavy et al. 2009; Struck et al. 2011),
nuchal organs have likely been lost in Clitellata (Purschke et al.
2000), as well as in Echiura, Myzostomida, and Siboglinidae,
which also lack nuchal organs. The presence of nuchal organs
in Sipuncula is controversially discussed (Purschke 1997) and
requires ultrastructural investigations in more species. On the
other hand, the lack of nuchal organs in Oweniidae and
Magelonidae is most likely a primary absence given the results
presented herein. Thus, nuchal organs are likely not an autapomorphy of Annelida (Purschke 2002).
Therefore, according to our analyses, the ancestral annelid
possessed a pair of either sensory or grooved palps, bicellular
eyes, biramous parapodia bearing simple chaeta, and lacked
nuchal organs (fig. 3, supplementary table S7, Supplementary
Material online). Eventually, characters such as the collageneous cuticle (Purschke 2002), the specific arrangement of
the body wall musculature (Purschke 2002), and the intraepidermal position of the nervous system (e.g., as found in
Oweniidae) have to be considered as part of the annelid
ground pattern as well.
Implications on the Annelid Radiation from the Fossil
Record
Investigating the fossil record, the oldest annelid fossils are
described for Sipuncula from the Lower Cambrian, which
MBE
Base of the Annelid Tree . doi:10.1093/molbev/msu080
FIG. 3. Ancestral reconstructions of body and parapodial characters of the last common ancestor of Annelida. (A) Annelida, derived from using the
parsimony and likelihood reconstruction option. (B) Annelida, derived from Struck et al. (2011). Body characters are shown on the left and parapodial
characters on the right. Question marks indicate that the state of the character is uncertain. bie, bicellular eyes; doc, dorsal cirrus; grp, grooved palps; isc,
internalized supporting chaetae; nuo, nuchal organ; sic, simple chaetae; sop, solid palps; vec, ventral cirrus.
seem remarkably similar to recent sipunculids (Huang et al.
2004). All three specimens were discovered in the
Maotianshan Shales and illustrate that the morphological appearance of Sipuncula has only slightly changed in the past
520 My. Polychaete fossils from the Lower Cambrian are
sparsely known. Although Maotianchaeta fuxianella and
Facivermis yunnanicus known from the Maotianshan Shales
resemble worm-like organisms (Li et al. 2007), a close relationship with lobopods seems more plausible (Liu et al. 2006).
Despite that, recently described polychaete fossils from the
Sirius Passet (518–505 Ma) were hypothesized as stem annelids (Morris and Peel 2008; Vinther et al. 2011). In addition,
various forms of tube-like fossils have been discovered from
the Lower and Middle Cambrian, which were presumably
constructed by stem group annelids (Skovsted and Peel
2011). Skovsted and Peel (2011) concluded that these fossils
had a similar life style as recent Chaetopteridae, based on the
absence of attachment structures, vertical orientation of the
tubes in the sediment, and opening on both ends, which
allowed the tube-dwelling worm-like animal to move and
filter water. However, these interpretations have to be considered with caution, because no specimen within the tubes
has been discovered yet. Nonetheless, the recovered phylogeny and the fossil record of Annelida indicate that lineages
leading to recent annelid groups such as Magelonidae +
Owenidae, Chaetopteridae, and Amphinomidae are as old
as Sipuncula. Consequently, these lineages are separated
since the Early Cambrian from the rest of Annelida.
Considering the age of the early annelid radiation and that
since this time several mass extinctions are recorded, including the end-Permian eradication of 90% of all marine species
(Jin et al. 2000), it might come as no surprise that we observe a
patchwork of morphologies and life modes of recent taxa
branching off from the basal part of the annelid tree. No
annelid taxon shows all ancestral conditions but is a composition of plesiomorphic and apomorphic characters even in
the case that the taxon is sister to all other annelids (Crisp and
Cook 2005). The increased taxon sampling of annelid lineages
clearly improved our picture of annelid evolution. The backbone presented in Struck et al. (2011) was largely supported
and some more lineages branching in the basal part of the
tree were discovered. Additional inclusion of so far lacking
annelid families in future phylogenomic studies will be an
important step in the refinement of our view of character
evolution in annelids. On the basis of our analyses, we strongly
advocate the establishment of additional annelid model
systems of species within the basal radiation, which would
contribute significantly to the understanding of the annelid
ground pattern.
Materials and Methods
Sampling, Transcriptome Library Construction, and
Sequencing
Supplementary table S1, Supplementary Material online, provides information on collection of organisms. All samples
were snap-frozen in liquid nitrogen, fixed immediately in
Ambion RNAlater (Life Technologies, Carlsbad, CA), or kept
in a seawater tank until RNA was extracted. For all species,
total RNA was extracted using Trizol Reagent (Life
Technologies, Carlsbad, CA) and purified with the RNeasy
Mini Kit (Qiagen, Hilden, Germany). Purification of mRNA
was performed using the Dynabeads mRNA Purification Kit
(Life Technologies, Carlsbad, CA). Subsequent fragmentation
was carried out using the Ambion RNA Fragmentation
Reagent to obtain fragments around 200–250 bp. Firststrand synthesis of cDNA was conducted with SuperScript
II reverse transcriptase (Life Technologies, Carlsbad, CA) using
random hexamer primers followed by second-strand synthesis using DNA Polymerase I and RNase H (Life Technologies,
Carlsbad, CA). Sequencing libraries were prepared following
the protocol of Meyer and Kircher (2010) using double indices
1397
MBE
Weigert et al. . doi:10.1093/molbev/msu080
as published in Kircher et al. (2012). The concentration and
purity of RNA, cDNA, and sequencing libraries was determined with Nanodrop and additionally on a Bioanalyzer
2100 (Agilent Technologies, CA). Most libraries were either
sequenced on the Illumina Genome Analyzer IIx or on the
Illumina MiSeq with 76 cycles paired end following the manufacturer’s protocols. Libraries of M. berkeleyi, Paramphinome
jeffreysi, and Phyllochaetopterus sp. were sequenced following
the standard Tru-Seq Illumina protocols and sequenced as
100 bp paired end runs on a Illumina Hi-Seq 2000. Magelona
berkeleyi and P. jeffreysi were run at Hudson Alpha Genomics
Service Lab (Huntville, AL) and Phyllochaetopterus sp. was
sequenced at the Emory Genetics Laboratory (Decatur, GA).
Libraries for Ophelia rathkei and Eurythoe complanata were
generated at GeneCore (EMBL, Heidelberg, Germany) following the standard protocol from Illumina for sequencing of
mRNA and sequenced on the Illumina Genome Analyzer
IIx with 105 cycles paired end. All sequence data were deposited in the National Center for Biotechnology Information
(NCBI) sequence read archive.
Processing Raw Data and Sequence Assembly
Bases were called with IBIS 1.1.2 (Kircher et al. 2009), adaptor
and primer sequences were removed, and reads with low
complexity and false paired indices were discarded. Raw
data of all libraries were trimmed by applying a filter of 15,
discarding all reads with more than five bases below a quality
score of 15. The quality of all sequences was checked using
FastQC (http://www.bioinformatics.babraham.ac.uk/projects/
fastqc/, last accessed March 6, 2014). Subsequently all libraries,
except libraries of O. rathkei and E. complanata, were assembled de novo using the CLC Genomics Workbench 5.1 (CLC
bio, Århus, Denmark) with the following settings: mismatch
cost 3; insertion cost 3; deletion cost 3; length fraction 0.5;
similarity fraction 0.8; minimum contig length 200; automatic
word size; automatic bubble size; and contig adjustment by
mapped reads. Data used for the assembly of Myzostoma
cirriferum comprised reads which were generated previously
(supplementary table S1, Supplementary Material online) and
reads of an additional run of the identical library on the
Illumina Genome Analyzer IIx with 76 cycles paired end.
Libraries of O. rathkei and E. complanata were assembled
using Velvet v. 1.1.04 (Zerbino and Birney 2008) and OASES
v. 0.1.21 (Schulz et al. 2012) with the default parameters. All
assembled libraries were checked for possible (cross) contamination by local Blast with 18S and several putatively single
copy ribosomal proteins and were rechecked on NCBI.
Obtaining and Processing Data from Other Taxa
Additional data for 36 annelids, 3 nemerteans, 4 molluscs, 3
brachiopods, 2 phoronids, 2 bryozoans, 2 entoprocts, and 1
cycliophoran were obtained from public resources of NCBI
(run by the National Institutes of Health), including the ESTs
database, the Sequence Read Archive, and the Trace archive.
Data for Capitella teleta, Helobdella robusta, and Lottia gigantea were extracted from the Joint Genome Institute (supplementary table S2, Supplementary Material online). Raw
1398
Illumina data were assembled with CLC Workbench 5.1
using identical parameters as described earlier. The 454 data
were assembled with MIRA v. 3.4.0 (http://www.chevreux.org,
last accessed March 6, 2014) using the default settings. Data
obtained from Struck et al. (2011) and all outgroup taxa
except Hanleya sp. and Lingula anatina were processed as
described in Hausdorf et al. (2007).
Orthology Assignment
Orthology prediction was performed using HaMStR version
8b with the representative option (Ebersberger et al. 2009).
The applied core-ortholog set lophotrochozoa_hmmer3,
which was generated using proteomes from seven primer
taxa (H. robusta, C. teleta, L. gigantea, Schistosoma mansoni,
Daphnia pulex, Apis mellifera, and Caenorhabditis elegans)
and comprises 2,339 orthologous genes, is deposited in data
dryad (http://datadryad.org/resource/doi:10.5061/dryad.g2qp5,
last accessed March 6, 2014). Each potential candidate
ortholog was then checked with reciprocal Blast against the
reference taxon H. robusta and was discarded if it did not
match to the expected ortholog from the reference taxon.
Redundant sequences were eliminated using a custom Perl
script, which checked for the presence of redundant sequence
identifiers in the assigned orthologous genes for each taxon.
Additionally, we checked our putative orthologous sequences
for contamination with protist sequences using local Blast
against the Apicomplexa proteome. We chose Apicomplexa
for comparisons as gregarine parasites have been described
for several annelids. Orthologous sequences that were more
similar to Apicomplexa than to the reference taxon H. robusta
were discarded.
Sequence Alignment, Masking and Concatenating,
and Matrix Reduction
Three data matrices were generated for phylogenetic analyses:
data set 79 comprising 79 taxa; data set 77, which excludes the
long-branching myzostomids resulting in a matrix of 77 taxa;
and data set 72 comprising 72 taxa due to the exclusion of all
outgroups except Mollusca, Nemertea, and Brachiopoda (table
1). For all data sets, single gene alignments of each orthologous
gene were generated separately using MAFFT (Katoh et al.
2002), and gaps and highly diverse amino acid positions were
removed from the data matrix with REAP (Hartmann and
Vision 2008). All masked single gene alignments were concatenated into a supermatrix using a custom Perl script. To investigate the influence of missing data on the topology of the
tree, we generated different data subsets from all three data
sets. To accomplish that, we enhanced the information content by increasing matrix density using the program MARE
(MAtrix REduction), which examines phylogenetic signal as
assessed by quartet mapping (MARE v0.1.2-rc, http://www.
zfmk.de, last accessed March 6, 2014). Three different weighing
parameters ( = 1, 1.5, or 2) were applied to generate three
data subsets differing in gene coverage and number of genes
for the 77- and as well for the 79-taxa data sets (table 1),
resulting in six data subsets. For the 72-taxa data set, only the
weighting parameter = 2 was applied. To retain all taxa
MBE
Base of the Annelid Tree . doi:10.1093/molbev/msu080
within a given data set, the parameter –c was applied for all
species. In total, seven data subsets were generated (table 1).
For all data subsets generated by MARE, base composition
heterogeneity and proportion of missing sequence data were
calculated for each taxon (table 1) using BaCoCa (Kück and
Struck 2014). To assess base composition heterogeneity, we
employed taxon-specific RCFV values that determine the absolute deviation of the base composition of a taxon from the
mean across all taxa (Zhong et al. 2011). All alignments are
available at data dryad (http://datadryad.org/resource/doi:10.
5061/dryad.g2qp5, last accessed March 6, 2014).
Phylogenetic Analyses and Testing
ProtTest2.4 was used to select the best fitting amino acid
substitution model (Abascal et al. 2005), suggesting the
LG + + I model. For all seven data subsets, maximum likelihood analyses were conducted with RAxML v. 7.3.1
(Stamatakis 2006). Rapid BS was applied with 500 bootstrap
replicates each under the CAT approximation, and searches
for the best tree were performed 10 times for each data set.
Leaf stability indices were determined using Phyutility v. 2.2
(Smith and Dunn 2008) and long-branch taxa were identified
using the LB score implemented in TreSpEx v.b042 (Struck
2013, www.annelida.de, last accessed March 6, 2014). The LB
score determines the percentage deviation of the average
pair-wise patristic distance of a taxon to all other taxa in
the tree relative to the average pair-wise patristic distance
of all taxon to each other. Thus, this score is a tree-based
measurement, which is independent of the root of the tree
in contrast to tip-to-root measurements. To test whether the
best trees differs significantly from different a priori hypotheses mainly based on morphology (supplementary table S3,
Supplementary Material online), per-site log likelihoods of the
best tree from the unconstrained and constrained analyses of
the data set comprising 77 taxa and MARE settings of = 1/
1.5/2 were computed using RAxML v. 7.3.1 (Stamatakis 2006).
For hypothesis testing, an AU test was conducted using
CONSEL (Shimodaira and Hasegawa 2001). Moreover, differences in psL of each constrained analysis in comparison to
the best tree were also used to explore if the presence of
paralogous or xenologous sequences were responsible for
the topological differences observed in the two compared
trees (Smith et al. 2011; Struck 2013). To make this assessment, psL was plotted against amino acid positions to determine whether the phylogenetic signal for each topology
was present across partitions in the alignment or concentrated into a single or few genes (data dryad: files S1–S9).
Signal present in just a few genes indicates follow-up analyses
maybe warranted to identify paralogy or xenology.
Ancestral Character State Reconstruction
Ancestral state reconstructions using both the likelihood and
the parsimony criterion were conducted as described in
Struck et al. (2011) based on a family tree derived from the
trees shown in supplementary figure S1, Supplementary
Material online. The morphological data matrix from the
study of Struck et al. (2011) was expanded to include
Acrocirridae,
Eunicidae,
Magelonidae,
Nephytidae,
Oweniidae, Polynoidae, Sabellaridae, Sabellidae, and
Tomopteridae. Character states were based on Zrzavy et al.
(2009) but slightly modified by updating/changing the coding
of characters related to “shape of parapodia,” “pygidial cirri,”
“uncini,” “hooks,” and “presence of eyes” according to Struck
et al. (2011) to ensure comparability with the results of the
study of Struck et al. (2011) (data dryad: files S10 and S11).
Supplementary Material
Supplementary tables S1–S7 and figures S1–S7 are available at
Molecular Biology and Evolution online (http://www.mbe.
oxfordjournals.org/).
Acknowledgments
The authors thank Marie-Theres Gansauge for technical assistance and Anne-C. Zakrzewski and Harald Hausen for collecting specimens of Spiochaetopterus sp. Special thanks to
Natascha Hill and Alexander Donath for the help with installing the HaMStR software and with the Perl scripts used in the
pipeline. The authors are also grateful to Tomas Larsson,
Michael Gerth, and Lars Hering for help in data processing
and suggestions and Carlos Sanchez Ortiz for providing us a
picture of Eurythoe complanta var. mexicana. They thank the
“Station Biologique de Roscoff” for specimen supply and providing of laboratory facilities. This study was supported by
DFG grants (STR-683/7-1, STR-683/8-1, BL787/5-1, and in
part HA 2763/5, Pu84/6-2, Pu84/6-3), the National Science
Foundation USA (DEB-1036537), and the EU due to
ASSEMBLE grant agreement no. 227799 (http://www.
assemblemarine.org).
References
Abascal F, Zardoya R, Posada D. 2005. ProtTest: selection of best-fit
models of protein evolution. Bioinformatics 21:2104–2105.
Bartolomaeus T. 1995. Secondary monociliarity in the Annelida: monociliated epidermal cells in larvae of Magelona mirabilis (Magelonida).
Microfauna Mar. 10:327–332.
Bleidorn C. 2007. The role of character loss in phylogenetic reconstruction as exemplified for the Annelida. J Zool Syst Evol Res. 45:299–307.
Bleidorn C, Eeckhaut I, Podsiadlowski L, Schult N, McHugh D, Halanych
KM, Milinkovitch MC, Tiedemann R. 2007. Mitochondrial genome
and nuclear sequence data support Myzostomida as part of the
annelid radiation. Mol Biol Evol. 24:1690–1701.
Bleidorn C, Podsiadlowski L, Zhong M, Eeckhaut I, Hartmann S,
Halanych KM, Tiedemann R. 2009. On the phylogenetic position
of Myzostomida: can 77 genes get it wrong? BMC Evol Biol. 9:150.
Bleidorn C, Vogt L, Bartolomaeus T. 2003. New insights into polychaete
phylogeny (Annelida) inferred from 18S rDNA sequences. Mol
Phylogenet Evol. 29:279–288.
Bubko OV, Minichev YS. 1972. Nervous system in Oweniidae
(Polychaeta). Zool Zhurnal. 51:1288–1299.
Bullock TH, Horridge GA. 1965. Structure and function in the nervous
system of invertebrates. San Francisco (CA): W. H. Freeman and
Company.
Capa M, Parapar J, Hutchings P. 2012. Phylogeny of Oweniidae
(Polychaeta) based on morphological data and taxonomic revision
of Australian fauna. Zool J Linn Soc. 166:236–278.
Clark RB. 1969. Systematics and phylogeny: Annelida, Echiura, Sipuncula.
In: Florkin M, Scheer BT, editors. Chemical zoology. Vol. 4. New York:
Academic Press. p. 1–68.
Crisp MD, Cook LG. 2005. Do early branching lineages signify ancestral
traits? Trends Ecol Evol. 20:122–128.
1399
Weigert et al. . doi:10.1093/molbev/msu080
Dordel J, Fisse F, Purschke G, Struck TH. 2010. Phylogenetic position of
Sipuncula derived from multi-gene and phylogenomic data and its
implication for the evolution of segmentation. J Zool Syst Evol Res.
48:197–207.
Ebersberger I, Strauss S, von Haeseler A. 2009. HaMStR: profile hidden
Markov model based search for orthologs in ESTs. BMC Evol Biol. 9:
157.
Eibye-Jacobsen D, Vinther J. 2012. Reconstructing the ancestral annelid.
J Zool Syst Evol Res. 50:85–87.
Fauchald K. 1977. The polychaete worms. Definitions and keys to the
orders, families and genera. Nat Hist Mus Los Ang. 28:1–188.
Fauchald K, Rouse G. 1997. Polychaete systematics: past and present.
Zool Scripta. 26:71–138.
Gardiner SL. 1978. Fine structure of the ciliated epidermis on the tentacles of Owenia fusiformis (Polychaeta, Oweniidae). Zoomorphologie
91:37–48.
Glaessner MF. 1976a. Early Phanerozoic annelid worms and their geological and biological significance. J Geol Soc. 132:259–275.
Glaessner MF. 1976b. A new genus of late Precambrian polychaete
worms from South Australia. Trans R Soc South Aust. 100:169–170.
Hartmann S, Helm C, Nickel B, Meyer M, Struck TH, Tiedemann R,
Selbig J, Bleidorn C. 2012. Exploiting gene families for phylogenomic
analysis of myzostomid transcriptome data. PLoS One 7:e29843.
Hartmann S, Vision TJ. 2008. Using ESTs for phylogenomics: can one
accurately infer a phylogenetic tree from a gappy alignment? BMC
Evol Biol. 8:95.
Hausdorf B, Helmkampf M, Meyer A, Witek A, Herlyn H, Bruchhaus I,
Hankeln T, Struck TH, Lieb B. 2007. Spiralian phylogenomics supports the resurrection of Bryozoa comprising Ectoprocta and
Entoprocta. Mol Biol Evol. 24:2723–2729.
Helm C, Bernhart SH, Siederdissen CHZ, Nickel B, Bleidorn C. 2012. Deep
sequencing of small RNAs confirms an annelid affinity of
Myzostomida. Mol Phylogenet Evol. 64:198–203.
Hermans CO. 1969. The systematic position of the Archiannelida. Syst
Biol. 18:85–102.
Huang DY, Chen JY, Vannier J, Salinas JIS. 2004. Early Cambrian sipunculan worms from southwest China. Proc R Soc B Biol Sci. 271:
1671–1676.
Jamieson BGM, Ferraguti M. 2006. Non-leech Clitellata. In: Rouse G,
Pleijel F, editors. Reproductive biology and phylogeny of Annelida.
Enfield (CT): Science Publishers. p. 235–392.
Jekely G, Colombelli J, Hausen H, Guy K, Stelzer E, Nedelec F, Arendt D.
2008. Mechanism of phototaxis in marine zooplankton. Nature 456:
395–399.
Jin YG, Wang Y, Wang W, Shang QH, Cao CQ, Erwin DH. 2000. Pattern
of marine mass extinction near the Permian-Triassic boundary in
South China. Science 289:432–436.
Katoh K, Misawa K, Kuma K, Miyata T. 2002. MAFFT: a novel method
for rapid multiple sequence alignment based on fast Fourier transform. Nucleic Acids Res. 30:3059–3066.
Kircher M, Sawyer S, Meyer M. 2012. Double indexing overcomes inaccuracies in multiplex sequencing on the Illumina platform. Nucleic
Acids Res. 40:e3.
Kircher M, Stenzel U, Kelso J. 2009. Improved base calling for the
Illumina Genome Analyzer using machine learning strategies.
Genome Biol. 10:R83.
Kristof A, Wollesen T, Wanninger A. 2008. Segmental mode of neural
patterning in sipuncula. Curr Biol. 18:1129–1132.
Kück P, Struck TH. 2014. BaCoCa—a heuristic software tool for the
parallel assessment of sequence biases in hundreds of gene and
taxon partitions. Mol Phylogenet Evol. 70:94–98.
Kvist S, Siddall ME. 2013. Phylogenomics of Annelida revisited: a cladistic approach using genome-wide expressed sequence tag data
mining and examining the effects of missing data. Cladistics 29:
435–448.
Li GX, Steiner M, Zhu XJ, Yang AH, Wang HF, Erdtmann BD. 2007. Early
Cambrian metazoan fossil record of South China: generic diversity
and radiation patterns. Palaeogeogr Palaeoclimatol Palaeoecol. 254:
229–249.
1400
MBE
Liu JN, Han J, Simonetta AM, Hu SX, Zhang ZF, Yao Y, Shu DG.
2006. New observations of the lobopod-like worm Facivermis
from the Early Cambrian Chengjiang Lagerstatte. Chin Sci Bull.
51:358–363.
McHugh D. 1997. Molecular evidence that echiurans and pogonophorans are derived annelids. Proc Natl Acad Sci U S A. 94:
8006–8009.
McHugh D. 2000. Molecular phylogeny of the Annelida. Can J Zool. 78:
1873–1884.
Meyer M, Kircher M. 2010. Illumina sequencing library preparation for
highly multiplexed target capture and sequencing. Cold Spring Harb
Protoc. 2010:pdb.prot5448.
Morris SC, Peel JS. 2008. The earliest annelids: lower Cambrian polychaetes from the Sirius Passet Lagerstatte, Peary Land, North
Greenland. Acta Palaeontol Pol. 53:135–146.
Mwinyi A, Meyer A, Bleidorn C, Lieb B, Bartolomaeus T, Podsiadlowski L.
2009. Mitochondrial genome sequence and gene order of Sipunculus
nudus give additional support for an inclusion of Sipuncula into
Annelida. BMC Genomics 10:27.
Nielsen C. 2002. The phylogenetic position of Entoprocta, Ectoprocta,
Phoronida, and Brachiopoda. Integr Comp Biol. 42:685–691.
Orrhage L, Müller MCM. 2005. Morphology of the nervous system of
Polychaeta (Annelida). Hydrobiologia 535:79–111.
Purschke G. 1997. Ultrastructure of nuchal organs in polychaetes
(Annelida)—new results and review. Acta Zool. 78:123–143.
Purschke G. 1999. Terrestrial polychaetes—models for the evolution of
the Clitellata (Annelida)? Hydrobiologia 406:87–99.
Purschke G. 2002. On the ground pattern of Annelida. Organ Divers Evol.
2:181–196.
Purschke G, Hessling R, Westheide W. 2000. The phylogenetic position
of the Clitellata and the Echiura—on the problematic assessment of
absent characters. J Zool Syst Evol Res. 38:165–173.
Purschke G, Jouin C. 1988. Anatomy and ultrastructure of the ventral
pharyngeal organs of Saccocirrus (Saccocirridae) and Protodriloides
(Protodriloidae fam. n.) with remarks on the phylogenetic relationships within the Protodrilida (Annelida: Polychaeta). J Zool. 215:
405–432.
Retallack GJ. 2007. Growth, decay and burial compaction of Dickinsonia,
an iconic Ediacaran fossil. Alcheringa 31:215–240.
Rieger RM. 1976. Monociliated epidermal cells in Gastrotricha: significance for concepts of early metazoan evolution. Z Zool Syst Evol. 14:
198–226.
Rieger RM. 1988. Comparative ultrastructure and the Lobatocerebridae:
keys to understand the phylogenetic relationship of Annelida and
the acoelomates. Microfauna Mar. 4:373–382.
Rouse GW. 1999. Trochophore concepts: ciliary bands and the evolution
of larvae in spiralian Metazoa. Biol J Linn Soc. 66:411–464.
Rouse GW, Fauchald K. 1997. Cladistics and polychaetes. Zool Scripta. 26:
139–204.
Rousset V, Pleijel F, Rouse GW, Erseus C, Siddall ME. 2007. A molecular
phylogeny of annelids. Cladistics 23:41–63.
Schulz MH, Zerbino DR, Vingron M, Birney E. 2012. Oases: robust de
novo RNA-seq assembly across the dynamic range of expression
levels. Bioinformatics 28:1086–1092.
Schulze A, Cutler EB, Giribet G. 2005. Reconstructing the phylogeny of
the Sipuncula. Hydrobiologia 535:277–296.
Seaver EC. 2003. Segmentation: mono- or polyphyletic? Int J Dev Biol. 47:
583–595.
Shimodaira H, Hasegawa M. 2001. CONSEL: for assessing the confidence
of phylogenetic tree selection. Bioinformatics 17:1246–1247.
Siddall ME, Bely AE, Borda E. 2006. Hirudinida. In: Rouse G, Pleijel F,
editors. Reproductive biology and phylogeny of annelida. Enfield
(CT): Science Publishers. p. 392–429.
Skovsted CB, Peel JS. 2011. Hyolithellus in life position from the lower
Cambrian of North Greenland. J Paleontol. 85:37–47.
Smart TI, Von Dassow G. 2009. Unusual development of the mitraria
larva in the polychaete Owenia collaris. Biol Bull. 217:253–268.
Smith SA, Dunn CW. 2008. Phyutility: a phyloinformatics tool for trees,
alignments and molecular data. Bioinformatics 24:715–716.
Base of the Annelid Tree . doi:10.1093/molbev/msu080
Smith SA, Wilson NG, Goetz FE, Feehery C, Andrade SCS, Rouse GW,
Giribet G, Dunn CW. 2011. Resolving the evolutionary relationships
of molluscs with phylogenomic tools. Nature 480:364–367.
Sperling EA, Vinther J, Moy VN, Wheeler BM, Semon M, Briggs DEG,
Peterson KJ. 2009. MicroRNAs resolve an apparent conflict between
annelid systematics and their fossil record. Proc R Soc B Biol Sci. 276:
4315–4322.
Stamatakis A. 2006. RAxML-VI-HPC: maximum likelihood-based phylogenetic analyses with thousands of taxa and mixed models.
Bioinformatics 22:2688–2690.
Storch V. 1968. Zur vergleichenden Anatomie der segmentalen
Muskelsysteme und zur Verwandtschaft der Polychaeten-Familien.
Z Morphol Tiere. 63:251–342.
Struck TH. 2011. Direction of evolution within Annelida and the definition of Pleistoannelida. J Zool Syst Evol Res. 49:340–345.
Struck TH. 2013. The impact of paralogy on phylogenomic studies—a
case study on annelid relationships. PLoS One 8:e62892.
Struck TH, Nesnidal MP, Purschke G, Halanych KM. 2008. Detecting
possibly saturated positions in 18S and 28S sequences and
their influence on phylogenetic reconstruction of Annelida
(Lophotrochozoa). Mol Phylogenet Evol. 48:628–645.
MBE
Struck TH, Paul C, Hill N, Hartmann S, Hosel C, Kube M, Lieb B, Meyer A,
Tiedemann R, Purschke G, et al. 2011. Phylogenomic analyses unravel annelid evolution. Nature 471:95–98.
Struck TH, Schult N, Kusen T, Hickman E, Bleidorn C, McHugh D,
Halanych KM. 2007. Annelid phylogeny and the status of
Sipuncula and Echiura. BMC Evol Biol. 7:57.
Vinther J, Eibye-Jacobsen D, Harper DAT. 2011. An Early Cambrian stem
polychaete with pygidial cirri. Biol Lett. 7:929–932.
Westheide W. 1997. The direction of evolution within the Polychaeta.
J Nat Hist. 31:1–15.
Wilson DP. 1982. The larval development of three species of Magelona
(Polychaeta) from localities near Plymouth. J Mar Biol Ass UK. 62:
385–401.
Zerbino DR, Birney E. 2008. Velvet: algorithms for de novo short read
assembly using de Bruijn graphs. Genome Res. 18:821–829.
Zhong M, Hansen B, Nesnidal M, Golombek A, Halanych KM, Struck
TH. 2011. Detecting the symplesiomorphy trap: a multigene phylogenetic analysis of terebelliform annelids. BMC Evol Biol. 11:369.
Zrzavy J, Riha P, Pialek L, Janouskovec J. 2009. Phylogeny of Annelida
(Lophotrochozoa): total-evidence analysis of morphology and six
genes. BMC Evol Biol. 9:189.
1401