Introduction
Attempts to explain the origin of metazoan life seek to unravel both the transition
from (1) single-celled to multicellular organisms and (2) diploblastic to triploblastic
body plans. The most favored scenarios are based on five well-known hypotheses on
the “urmetazoon” bauplan: Haeckel's gastraea, Jägersten's bilaterogastraea, Metschnikoff's
phagocytella, Lankester's planula, and Bütschli's placula [1–5]. Attempts to unravel
the urmetazoon bauplan and to provide support for any of the five hypotheses depends
on identifying the most basal extant diploblast group. Two phylogenetic alternatives
have remained under discussion; one sees the sponges (Porifera) and the other the
placozoans (Placozoa) as basal relative to all other diploblast groups [6–10]. The
latter view was accepted for the most part of the last century. The presence of only
four somatic cell types, the smallest metazoan genome, and the lack of any foot or
head structures, any anterior–posterior organization, or any kind of organs, and both
a basal lamina and an extracellular matrix (ECM) places Trichoplax in a basal and
isolated position relative to all other metazoan phyla [11–16] (cf. [17], however).
Tangled Roots at the Base of the Metazoan Tree of Life
Mainly because of misinterpretation of life cycle stages between Trichoplax adhaerens
and the hydrozoan Eleutheria dichotoma, Placozoa lost their predominant role as the
key model system for studying the origin of metazoan life [5,17]. This outcome was
nourished by molecular studies based on a variety of character sources, which created
a series of conflicting phylogenetic scenarios in which most often Porifera came out
basal [18–24]. Figure 1 shows six plausible scenarios for the relationships of five
taxonomic groups (Bilateria, Cnidaria, Ctenophora, Porifera, and Placozoa) and two
plausible arrangements for four taxa when Placozoa are left out that are critical
in assessing the early relationships of metazoans. For five taxa and one outgroup,
there are 105 ways to arrange these taxa in dichotomous branching trees. Nearly 95%
of these possible trees can be eliminated as not plausible based on existing data.
All six of the hypotheses in Figure 1 have been suggested as viable in the literature
over the past two decades (see Table S1 for a summary of papers in the last decade
addressing the phylogenetics of these taxa).
Figure 1
Discussed Relationships at the Base of the Metazoan Tree
Potential arrangements of five critical taxa (B, Bilateria; Cn, Cnidaria; Ct, Ctenophora;
P, Placozoa; and S, Porifera) are shown on the right, and some hypotheses in the literature
with only four taxa (Placozoa omitted) on the left. Arrows indicate the root of the
networks. The letters at the arrows are for reference to Table S1. The uppercase letters
refer to publications in Table S1 that support the indicated root for trees without
Placozoa. The lowercase letters refer to publications in Table S1 that support the
root for trees with all five taxa.
All six hypotheses have been suggested in publications in the last year alone. For
instance, Srivastava et al. (2008) [23] hypothesize Placozoa as the sister group to
both Cnidaria and Bilateria, with sponges branching off earlier (arrow b in Figure
1). Another recent study, which suggests a basal position for Ctenophora and Anthozoa
(arrow E in Figure 1), unfortunately does not add to the issue, since it does not
include Placozoa in the analysis [25]. However, this study does suggest that Cnidaria
are not sister to Bilateria, but rather to Porifera [25]. A study that does include
Placozoa [26] also suggests that Bilateria and Placozoa are basal metazoans (arrow
a in Figure 1). Striking examples of the diversity of hypotheses generated on these
taxa are recent analyses of mitochondrial genome sequence data [27–29] that place
Bilateria as sister to all non-Bilateria, with Placozoa as the most basal diploblast
(arrow e in Figure 1). In the following, we use the term “diploblasts” for all nonbilaterian
metazoans; we do not intend to contribute to the discussion of whether diploblastic
animals may have a mesoderm, however [1,30–33].
Results and Discussion
A Concatenated Dataset for Metazoa
Given that both nonphylogenetic interpretation of morphological data as well as molecular
analyses of sequence data have failed to resolve the issue, a more comprehensive,
systematic analysis of morphological data and new molecular markers are now a requisite
for identifying the root of the metazoan tree of life. To approach this goal, we conducted
concatenated analyses for 24 metazoan taxa from all of the major organismal lineages
in this part of the tree of life that included morphological characters (17 characters),
both mitochondrial and nuclear ribosomal gene sequences (five gene partitions for
6,111 nucleotide positions) and molecular morphology [8] (ten characters), as well
as nuclear coding genes (16 gene partitions derived from our database searches and
another 18 gene partitions derived from the Dunn et al. (2008) study [25]; see Materials
and Methods) for 8,307 amino acid positions and protein coding genes (16 gene partitions
for 3,004 amino acid characters) to resolve phylogenetic relationships between recent
diploblast groups. The total number of characters included was 17,664 from 51 partitions,
giving 7,822 phylogenetically informative characters. We also constructed a matrix
with a larger number of taxa based on the Dunn et al. (2008) [25] study with 73 taxa
for the same gene partitions (see Materials and Methods and Tables S2 and S4). This
matrix had 17,637 total characters and 9,421 phylogenetically informative characters.
In addition, Hox gene expression was compared for a placozoan and a cnidarian bauplan
to test predictions from the placula hypothesis [5].
Clarity and Confusion at the Root of the Metazoan Tree
Parsimony, likelihood (with morphological characters removed), and mixed Bayesian
analysis of the smaller concatenated matrix using a variety of approaches, weighting
schemes, and models is generally consistent with the view that Bilateria and diploblasts
(Porifera, Ctenophora, Placozoa, and Cnidaria) are sister groups. In addition, Placozoa
are robustly observed as the most basal diploblast group (Figure 2 and Figure 3).
Figure 3 shows the support for several hypotheses of monophyly obtained from diverse
methods of analysis. Porifera, Bilateria, and Fungi all form strong monophyletic groups
(Figure 3). The four cnidarian classes (Anthozoa, Hydrozoa, Scyphozoa, and Cubozoa)
together with the Ctenophora form a monophyletic group, the “Coelenterata.” Within
the Cnidaria, the generally accepted basal position of the anthozoans is also recovered
by this analysis [34,35]. Both choanoflagellates and Placozoa are strongly excluded
from a Porifera–Coelenterata monophyletic group. The basal position of Placozoa is
also strongly supported by comparing the phylogeny in Figure 2 with hypotheses that
place it more derived, using the statistical approach of Shimodaira and Hasegawa [36,37].
This battery of tests (Table 1) demonstrates that the basal position of the Placozoa
is significantly better than other hypotheses. The 95% confidence tree includes the
Maximum Likelihood (ML) and Bayesian trees (both with Placozoa as basal in the diploblasts)
with a cumulative expected likelihood weight (ELW) of 0.960763.
Figure 2
Maximum Likelihood Phylogenetic Tree of Metazoan Relationships Using the Concatenated
Data Matrix
Node support is based on the best ML tree filtered through 1,000 rapid bootstrap replicates.
Only support values below 100% are shown. Bayesian inference supported strongly (posterior
probability = 1.0) all nodes with the exception of monophyly of Cnidaria. The maximum
a posteriori and the Bayesian 50% majority-rule consensus trees disagreed with the
best ML tree in supporting a Ctenophora–Anthozoa clade with posterior probability
of 0.98. Please note that “Coelenterata” is not a taxonomic unit, but rather it is
a traditional grouping for reasons of convenience. The alpha shape parameters of the
Gamma distribution were 0.507454 and 0.651659 for the nucleotide and amino acid partitions,
respectively. Log-likelihood = −261429.821426.
Figure 3
Phylogeny of Animals and Weighting Schemes
The impact of several weighting schemes on the phylogenetic hypothesis in Figure 2.
The values in the table are jackknife values for maximum parsimony, rapid bootstrap
for ML, and posterior clade probabilities for Bayesian inference. The color coding
for the values is shown at the bottom of the table. The major monophyletic groups
examined for jackknife support in Figure 2 are indicated in the top row. See Figure
2 for nodes defined by these groups. Monosiga refers to placing Monosiga as basal
to Metazoa, and Placozoa refers to placing Placozoa as basal to diploblasts. Total
in the first row refers to the entire dataset analyzed with equal weighting of all
characters. The next four rows show results for analyses of partitioned datasets:
mtDNA, mitochondrial partition; Nuclear, nuclear; Protein, protein; and rRNA, ribosomal
RNAs from both nuclear and mitochondrial genomes. The bottom rows show results for
various weighting schemes; 2:rRNA, 10:rRNA, and 100:rRNA refer to weighting schemes
in which transversions are weighted 2, 10, and 100 times more than transitions, respectively.
Protein weighting schemes are Gonnet weighting matrix, Whelan and Goldman (WAG) matrix,
Le and Gascuel (LG) matrix, and genetic identity (GI). For details on weighting matrices,
see Figure S4.
Table 1
Comparison of Competing Phylogenetic Hypotheses
The tree topology shown in Figure 2 summarizes the best supported phylogenetic hypothesis
obtained by using Maximum Parsimony, ML, and Bayesian analyses of the concatenated
dataset. Analysis of the larger matrix (Figure S2) was less well resolved within the
Bilateria, but showed the same general topology as the smaller analysis. Specifically,
Bilateria are monophyletic and sister to the diploblasts, with the choanoflagellate
Monosiga basal to these taxa with high jackknife values and Bayesian posteriors. Diploblasts
are also monophyletic, and Placozoa are the most basal taxon in the diploblasts. In
addition, within the diploblasts, Porifera and Coelenterata are monophyletic, and
within Bilateria, Ecdysozoa and Deuterostomia are monophyletic; all groupings with
high node support.
The topology within the diploblasts is also robust when Bilateria are removed from
the analysis. The full analysis seemingly misplaces the Bilateria clade as the sister
to all diploblasts. The classical position of the Bilateria is in a highly derived
position from within the diploblasts and usually sister to the Cnidaria. The seemingly
“weird” prediction of a basal Bilateria from the present analysis has been observed
before in other studies (see Table S1). Several studies have addressed phylogenetic
problems specific to this region of the tree of life and have suggested that this
region of the tree will be inherently difficult to resolve. These studies suggest
that the compression of splitting events in this region renders the resolution of
these nodes with high support difficult, if not impossible [38–42]. These studies
have suggested that even large amounts of data might not resolve the problem. Other
studies have pointed to taxon sampling and modeling as a potential problem in resolving
this part of the tree of life [25,38–40]. Another problem is that the large number
of molecular phylogenetic approaches creates multiple and possibly the most short-lived
hypotheses in biology. The large repertoire of algorithms, models, and assumptions
sometimes produces a forest of trees from the same dataset (cf. [43]). Thus, tree-building
procedures are highly crucial and deserve particular attention if this region of the
tree of life is to be resolved [38].
Possible Swamping by Mitochondrial Data?
Our analyses provide strong evidence for a basal position of Placozoa relative to
other diploblasts, and thus agrees with the mitochondrial genome data analyses (as
indicated by arrow f in Figure 1; [27,28]). It is therefore important to examine whether
the mitochondrial signal swamps out the nuclear data, to rule out the possibility
that the topology we present in Figure 2 is biased by mitochondrial information. Figure
S1 addresses this problem and demonstrates that nuclear information contributes positive
support to 16 of the 21 nodes in the tree. Mitochondrial information contributes positive
support to only 15 out of 21 nodes. In addition, examination of the amount of hidden
support contributed by nuclear versus mitochondrial data (not shown) shows that the
majority of the hidden support comes from nuclear information. Both of these results
using partitioned support measures indicate that the addition of nuclear data does
not conflict with mitochondrial information and is indeed contributing positively
to the overall phylogenetic hypotheses
Resurrecting the “Placula”
Although the hypothesis in Figure 2 is in conflict with a recent analysis of coding
genes from whole genomes [23] as well as is in conflict with other studies (Table
S1), the scenario presented here is consistent with another set of studies and also
with one of the major urmetazoon hypotheses, the placula hypothesis (Figure 4). This
hypothesis fuels intriguing scenarios for the mechanisms and direction of anagenetic
evolution in Metazoa, and in the form presented here, it can illustrate the derivation
of Cnidaria and Bilateria from a placozoan-like ancestor. A basal position of Placozoa
relative to Cnidaria, and diploblasts sister to Bilateria are cum grano salis consistent
with several recent molecular phylogenetic analyses ([23,27] and this study) encouraging
us to reconsider the placula hypothesis in a modern light.
Figure 4
Modern Interpretation and Modification of the Placula Hypothesis of Metazoan Origin
Here, a nonsymmetric and axis-lacking bauplan (placula) transforms into a typical
symmetric metazoan bauplan with a defined oral–aboral or anterior–posterior body axis.
In the placula transformation, a primitive disk consisting of an upper and a lower
epithelium (lower row), which can be derived from a flattened multicellular protist,
forms an external feeding cavity between its lower epithelium and the substrate (second
row from bottom). The latter is achieved by the placula lifting up the center of its
body, as this is naturally seen in feeding Trichoplax (i.e., the two Trichoplax images
derive from a nonfeeding (first row) and feeding (second row) individual. If this
process is continued, the external feeding cavity increases (cross section, third
row) while at the same time the outer body shape changes from irregular to more circular
(see oral views). Eventually, the process results in a bauplan in which the formerly
upper epithelium of the placula remains outside (and forms the ectoderm) and the formerly
lower epithelium becomes “inside” (and forms the entoderm; upper row). This is the
basic bauplan of Cnidaria and Porifera. Three of the four transformation stages have
living counterparts in the form of resting Trichoplax, feeding Trichoplax, and cnidarian
polyps and medusae (right column).
The above-outlined transformation of a placula into a cnidarian bauplan involves the
development of a main body axis and a head region, which allows the invention of new
structures and organs for feeding. From a developmental genetics point of view, a
single regulatory gene would be required to control separation between the lower and
upper epithelium (three lower rows). If the above scenario were correct, the following
empirical data would be congruent with it. In the form of the putative ProtoHox/ParaHox
gene, Trox-2, in Trichoplax, we find a single regulatory gene, marks the differentiation
of an as yet undescribed cell type at the lower–upper epithelium boundary in Trichoplax
[46]. More than one regulatory gene would be required to organize new head structures
originating from the ectoderm–entoderm boundary of the oral pole (upper row). Quite
noteworthy, two putative descendents of the Trox-2 gene, Cnox-1 and Cnox-3, show these
hypothesized expression patterns (Diplox expression upper row; for simplicity, only
the ring for Cnox-1 expression is shown; see Figure S4 for expression patterns of
both genes and Jakob et al. [46,52] for details. Cnox-1 and Cnox-3 expression both
mark the ectoderm-entoderm boundary at the oral pole in the hydrozoan Eleutheria dichotoma.
Both genes are expressed in parallel in a ring-shaped manner at the tip of the manubrium,
with Cnox-3 being expressed more ectodermally and Cnox-1 being expressed more entodermally
(unpublished data).
The comparison of Hox/ParaHox-like gene expression patterns in Placozoa and Cnidaria
creates a new working hypothesis for the origin of the entoderm, a main body axis,
and symmetry. Based on the undisputed evidence that Placozoa are basal relative at
least to Cnidaria, the Trox-2 gene is likely ancestral to Hox/ParaHox-like genes from
Cnidaria (as formerly suggested [44,45]). Trox-2 is expressed at the gastrodermis/epidermis
(lower/upper epithelium) boundary in Trichoplax [46]. Strikingly, we found similar
expression patterns for two putative Trox-2 descendents in the hydrozoan Eleutheria
dichotoma (Figure 4). These regulatory gene expression data mirror directly the beginning
and ending stage of a modern interpretation of the placula hypothesis. The latter
explains the origin of a symmetric bauplan with one or two defined body axes and an
internal feeding cavity from a simple placuloid (proto-placozoan–like) bauplan that
lacked all of the former characteristics. In the most parsimonious scenario, the expression
of a single regulatory gene defines polarity in Placozoa, i.e., the differentiation
of a lower versus upper epithelium. According to the proposed “new placula hypothesis,”
the nonsymmetric placozoan bauplan transforms into a symmetric Cnidaria (or also Bilateria)
bauplan by the former ring of epithelia boundary separation transforming into the
new “oral” region of the derived symmetric bauplan (Figure 4). This transformation
is simply the result of a placula lifting up its feeding epithelium in order to form
an external feeding cavity, keeping function and morphology of the epithelium unchanged.
In the final stage, the “oral” pole develops specialized organs, such as a mouth and
tentacles for feeding (cf. [47]). The latter could be driven by duplication of the
regulatory gene, which originally defined polarity in the placula (Figure 4; cf. [48]
for review). Observations on extant Placozoa and Cnidaria mirror this scenario almost
perfectly (Figure 4).
Although prediction and observation match nicely, one has to note, however, that no
gene or even gene family, no matter how important, can provide more than just indirect
support for a working hypothesis on a hypothetical animal bauplan that can never be
observed. It is important to note that multiple topologies can be consistent with
the placula hypothesis and that the form of the extant earliest-branching lineage
does not necessarily have to represent the form of the ancestor; we consider the latter,
however, the more parsimonious alternative. We also point out that the regulatory
gene family mentioned here, Hox/ParaHox-like genes, seems to be absent in sponges
[49]. A secondary loss of Hox/ParaHox-like genes in sponges seems plausible, and the
work by Peterson and Sperling, 2007 [50] provides some evidence for this assumption.
Whether a possible loss of a Hox/ParaHox gene might be related to the reduction of
epithelial organization in Porifera [3] remains an interesting speculation.
The Hox/ParaHox loss scenario in sponges is just one of several crucial questions
raised by the phylogeny in Figure 2. According to this phylogeny, diploblasts and
Bilateria both may have started from a placula-like bauplan as suggested in Figure
4 (“new placula hypothesis”). The shown new placula hypothesis illustrates a potential
transition from a nonsymmetric, axis-lacking placula into a radial symmetric and head–foot
axis organized cnidarian. In a similar way, the placula could also be transformed
into a Bilateria bauplan, i.e., a bilaterally symmetric bauplan with an anterior–posterior
body axis. One of the easiest models for adopting a bilateral symmetry suggests that
the “urbilaterian” kept the benthic lifestyle of the placula but adopted directional
movement. The latter almost automatically leads to an anterior–posterior and ventral–dorsal
differentiation. The pole moving forward develops a head and becomes anterior, the
body side facing the ground carries the mouth and thus by definition becomes ventral.
According to the above scenario, the main body axes of diploblastic animals and Bilateria
were independent inventions. Whereas an independent evolution of body axes in diploblastic
animals and Bilateria seems easily plausible, the independent evolution of other characters
(e.g., the nervous system; see below) seems less plausible given our knowledge of
the development and morphology of these characters.
We will never observe the hypothetical placula, but we may draw some conclusions from
Placozoa, which seem to have retained many of the characteristics of the placula if
our interpretation is valid. This scenario draws into question several aspects of
animal evolution that will require reinterpretation if this hypothesis is correct.
Most notable of these aspects is the evolution of the nervous system, which in the
hypothesis in Figure 2, can only be explained by convergent evolution of Cnidaria
and Bilateria nervous system organization. According to the placula hypothesis, we
suggest that the placula already had the genetic capability and basic building blocks
to build a nervous system, and that from here, the final build-up of the nervous system
developed via independent, but parallel, pathways in diploblasts and Bilateria. The
genome of the placozoan Trichoplax adhaerens indeed delivers some notable evidence
that the genetic inventory may precede morphological manifestation of organs [23].
For example, the placozoan genome harbors representatives of all major genes that
are involved in neurogenesis in higher animals, whereas placozoans show not the slightest
morphological hint of nerve or sensory cells. Quite noteworthy, however, is that placozoans
are quite capable of stimuli reception and perception used to coordinate behavioral
responses. In this light, the generally accepted unlikely convergent evolution of
a nervous system only looks unlikely from a morphological, but not from a genetic
and physiological, point of view.
Regardless of the need for reinterpretation of this and other anatomical characters,
the findings presented here provide a viable hypothesis for the major cladogenetic
events during the metazoan radiation. Given the basal position of Placozoa, we suggest
that at least for diploblastic metazoan life, the body plan started with the following:
an asymmetric body plan, a most simple morphology (only two steps above basic definition
[51]), a single ProtoHox gene, a large mitochondrial (mtDNA) genome, an outer feeding
epithelium that gave rise to the entoderm, and the smallest of all known (not secondarily
reduced) metazoan genomes. If the placula is also the ancestral state for metazoans
(i.e., the common ancestor of Bilateria and diploblasts in Figure 2), then the same
could be said for the urmetazoon.
Materials and Methods
Cloning and sequencing of target genes.
In order to extend the analyses of Rokas et al. [42] to basal metazoans also, we isolated
13 of the suggested target genes that were missing from the placozoan Trichoplax adhaerens.
These genes could be amplified by using the primer sets that had worked in the previous
study in sponges: TOA04, 05, 06, 09, 10, 11, 13, 15, 16, 17, 21, 25, 33, 48, 53, 56,
57, 59, 62, 65, 67, and 68. In order to obtain sequences of these genes for Placozoa
and to characterize variation within Placozoa, we also isolated six of these genes
from a second, distantly related placozoan species (Placozoa sp. H2, TunB clone, Tunisia).
For cubozoans, we filled gaps in the matrix by isolating three target genes from Carybdea
marsupialis (Table S5). We amplified target genes from cDNA. For both placozoan species,
some 200 healthy growing vegetative animals of each species were used for the isolation
of total RNA. Before extraction, animals were washed three times with sterile 3.5%
artificial seawater (ASW) and starved overnight to prevent algae contamination. Animals
were lysed in 500 μl of fresh homogenization buffer (HOM: 50 mM Tris HCl, 10 mM EDTA,
100 mM NaCl, 2.5 mM DTT, 0.5% SDS, 0.1% DEPC in ultrapure water [Gibco]; pH 8.0).
After addition of 25 μg of DEPC-treated Proteinase K, samples were stored for 30 min
at 65 °C. The homogenate was squeezed through a needle connected to a 2.5-ml syringe.
This protocol significantly increased RNA yield compared to conventional RNA extraction
kits. Nucleic acids were isolated by two rounds of phenol/chloroform/isoamyl alcohol
(25:24:1) purification. Nucleic acids were dissolved in ultrapure water, and DNA was
digested with DNase I (Fermentas). Total RNA was used for cDNA transcription with
poly-T primers following the manufacturer's protocol (Invitrogen Superscript II Kit).
Target genes were amplified after initial denaturation (3 min at 94 °C) by 40 rounds
of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 75 s, followed by a final elongation
step (5 min at 72 °C) using the Bioline Taq system following the manufacturer's recommendations
(Bioline). Amplified fragments of the predicted size were purified and cloned into
pGEM-T (Promega). Sequencing was performed on a Megabase 500 using the DYEnamic ET
Terminator Cycle Sequencing Kit (Amersham) or by using the service provided by Macrogen.
For further details, see Jakob et al. [46] and Table S5.
For a detailed explanation of the inclusion of sequences in the phylogenetic matrices
used in this study, see Table S2, which shows the source of sequences in this study.
We constructed two matrices, a small one composed of 24 taxa (see Figure 2) and a
large one composed of 73 taxa. For the smaller matrix, we chose nine bilaterian taxa
based on the availability of sequence information for a species. We chose three Lophotrochozoa,
three Ecdysozoa, and three Deuterostomia as representatives of the Bilateria. Other
ingroup taxa include representatives of the four classes of Cnidaria, the three major
groups of Porifera (Desmospongiae, Calcarea, and Hexactinellida), Placozoa, and Ctenophora.
Since rooting of the tree is critical, we attempted to break up the root by including
several outgroup species: two fungal species (Saccharomyces and Cryptococcus), Tetrahymena,
Trypanosoma, and Dictyostelium based on their relevance to the study and the availability
of genome-level information. Trypanosoma was used as outgroup species in all aspects
of the study, but the topology of resultant trees indicates that slime mold or Tetrahymena
could also be used. To increase the number of placozoan and cubozoan sequences, we
PCR amplified several genes as indicated in Table S5. Morphological characters were
scored for the taxa in this study as described in Schierwater and DeSalle (2007 [10];
see Table S3). Molecular “morphology” characters were also included for the taxa in
this study as scored by Ender and Schierwater, 2003 [8] (see Figure S3). The final
partitioned matrices for the smaller (24 taxa) and the larger (73 taxa) can be found
in Table S4. In addition to genes already available from whole mitochondrial sequencing
(15 genes) and nuclear genes (16 genes), we included 18 genes from the Dunn et al.
(2008) study [25]. These genes were chosen on the basis of taxonomic representation
being over 50% in the Dunn et al. (2008) study.
For the larger 73-taxon matrix, we included all of the taxa from the Dunn et al. (2008)
study (their smaller matrix in their Figure 2; [25]) plus Cubozoa, Scyphozoa, Placozoa,
Hexactinellida, Calcarea, Caenorhabditis, Tetrahymena, Trypanosoma, and Dictyostelium.
For this larger matrix, we filled in character information for these taxa for the
18 Dunn et al. (2008) [25] genes from GenBank as completely as possible. We used Blast
scores and existing annotations as criteria for assessing orthology for these added
sequences. In this larger matrix, we used only genes from the Dunn et al. (2008) study
[25] with greater than 50% taxon representation.
In situ hybridization and immunocytology.
RNA in situ hybridization studies were performed as described before [46,52]. For
immunocytology studies, polyclonal antibodies were produced to oligopeptides near
the C-terminal of the Trox-2, Cnox-1, and Cnox-3 proteins. For whole-mount analysis,
live animals were fixed for 1 h in 5% formaldehyde in sterile seawater. Immunocytochemistry
was performed with anti-Trox or anti-Cnox, respectively, antisera and goat anti-rabbit-AP
(Novagen) or FITC-conjugated goat anti-rabbit antibody (Sigma). Localization of antibody
complexes was revealed by staining with NBT and X-phosphate (Roche) or fluorescent
microscopy, respectively. Further details will be described elsewhere (S. Sagasser
et al. unpublished data).
Alignment.
To generate static alignments, we used MAFFT [53], initially with a gap opening penalty
of 1.5 and gap extension penalty of 0.123. We also examined the impact of varying
gap opening penalties by obtaining alignments using opening penalties of 1.0, 0.5,
and 0.1. The alteration of gap penalty only served to alter the number of characters
in our matrices and did not severely impact phylogenetic hypotheses.
Phylogenetic analysis.
For our 24-taxon matrix, we conducted parsimony, Bayesian, and likelihood analyses
as explained below. The 73-taxon matrix was analyzed with Bayesian inference. Phylogenetic
trees using static alignment were generated using PAUP v4b10 [54]. Tree searches were
accomplished using 1,000 random taxon additions and Tree Bisection Reconnection (TBR).
Jackknife measures for node support were obtained using PAUP with 30% character removal
and 1,000 repetitions. To examine the effect of character weighting in phylogenetic
analysis of this dataset, we implemented character weighting for nucleic acids and
amino acid partitions as follows. First, we implemented three schemes for weighting
transitions and transversions (100, 10, and 2) for nucleic acids. Second, we used
four transformation matrices for amino acid weighting: Gonnet [55], WAG [56], LG [57],
and Genetic Identity (GI). Bremer support measures (decay indices) [58], partitioned
Bremer and hidden support values [59,60] were generated using TreeRot v3 [61]. The
parallel implementation of MrBayes v3.1.2 [62,63] was used for Bayesian inference
of phylogeny. Two simultaneous runs with random starting trees were launched for two
million generations, each with a 1,000-step thinning, a 10% burn-in, and a temperature
parameter of 0.2 so as to lead to better mixing. All three data types (DNA, protein,
and morphology) were accommodated in the Bayesian analysis. We employed ML inference
in RAxML v7.0.4 [64] using the GTR substitution model for DNA [65,66] along with G-distributed
rate heterogeneity [67,68] and the Whelan and Goldman (WAG) amino acid substitution
matrix [55] with empirical residue frequencies coupled with G-distributed rate heterogeneity.
Node support was evaluated with 1,000 rapid bootstrap replicates [69]. Alternative
phylogenetic hypotheses were compared using the Shimodaira-Hasegawa test [37] and
expected likelihood weights [70], as implemented in RAxML.
Supporting Information
Figure S1
Positive or Negative Partitioned Bremer Support for All Nodes under Mitochondrial
versus Nuclear Gene Partitions
The shown analysis was done for one of the “plausible” parsimony trees. Other topologies
preferred by parsimony analysis gave similar inferences about support. The figure
shows whether the partitioned Bremer support values are positive negative or neutral.
This figure demonstrates that the nuclear versus mitochondrial partitions all provide
similar degrees of support for the various nodes in the tree. Note that over half
of the nodes acquire positive support from both partitions (11/21). Most of the negative
support in the tree is within the diploblast clade (six out of eight nodes) indicating
the instability of the relationships in this clade. Note also that the majority of
the negative support comes from mitochondrial partitions further strengthening our
contention that the mitochondrial partitions are NOT swamping the nuclear partitions.
Nodes at the base of the tree exhibit consistent support from all sources under the
shown partitioning scheme. Quite strikingly, nuclear proteins seem to provide the
highest positive support of all the characters in the analysis.
(70 KB PPT)
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Figure S2
Phylogenetic Tree for 73 Taxa Matrix with Bilateria Shown as Major Groups (A) and
Including All Taxonomic Names (B)
The 73 taxa are comprised of the 64 taxa from the Dunn et al. (2008) study [25] plus
nine taxa added from the present study. Since the topologies within Lophotrochozoa,
Ecdysozoa, and Deuterostomia are not discussed in our study, we have represented these
as major monophyletic groups in this figure (A).
All included taxa are listed in (B). The blue circles indicate that the support for
these nodes are 100% jackknife support for unweighted parsimony analysis and 1.0 posterior
Bayesian probability for parsmodel analysis in MrBayes. For four nodes relevant to
the present study from this larger analysis, the jackknife values and Bayesian posteriors
are listed next to the nodes, respectively.
(105 KB PPT)
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Figure S3
16S rRNA Secondary Structure Prediction
(126 KB PPT)
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Figure S4
In Situ Expression of Hox-Like Genes Cnox-1 and Cnox-3 in the Hydrozoan Eleutheria
dichotoma
The two Hox-like genes, Cnox-1 and Cnox-3, display differential spatiotemporal expression
patterns in the medusa stage. Cnox-1 (A1–A4) is expressed ectodermally in the so-called
Nesselring, an area of undifferentiated cells lining the ring canal of medusae (cross
section: A3, A4). Cnox-3 expression marks the most ectodermal oral part of the manubrium
(B1, B2). Staining is with NBT/X-phosphate (A1, B1) and fluorescein-labeled probes
(A2, B2); the scale bar indicates 50 μm. Pictures are reprinted from Jakob and Schierwater
(2007) [52].
(2.17 MB PPT)
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Table S1
Survey of the Literature for Hypotheses Concerning the Major Animal Lineages Discussed
in This Paper
(45 KB XLS)
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Table S2
GenBank Accession Numbers Used in This Study
(47 KB XLS)
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Table S3
Morphology Data Matrix
(24 KB DOC)
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Table S4
Alignment Matrix for 24 Taxa and 73 Taxa (in Nexus Format)
(1.70 MB TXT)
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Table S5
Disposition of PCR and Sequencing of Placozoan and Cubozoan Genes
(38 KB XLS)
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