Molecular Phylogenetics and Evolution 66 (2013) 551–557
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Review
Chasing the urmetazoon: Striking a blow for quality data? Hans-Jürgen Osigus a, Michael Eitel a,d, Bernd Schierwater a,b,c,⇑ a
ITZ, Division of Ecology and Evolution, Stiftung Tieraerztliche Hochschule Hannover, Germany American Museum of Natural History, New York, USA c Department of Ecology and Evolutionary Biology, Yale University, USA d The Swire Institute of Marine Science, Faculty of Science, School of Biological Sciences, The University of Hong Kong, Hong Kong b
a r t i c l e
i n f o
Article history: Available online 6 June 2012 Keywords: Urmetazoon Trichoplax Placozoa Non-bilaterian animals Placula hypothesis Metazoan evolution
a b s t r a c t The ever-lingering question: ‘‘What did the urmetazoan look like?’’ has not lost its charm, appeal or elusiveness for one and a half centuries. A solid amount of organismal data give what some feel is a clear answer (e.g. Placozoa are at the base of the metazoan tree of life (ToL)), but a diversity of modern molecular data gives almost as many answers as there are exemplars, and even the largest molecular data sets could not solve the question and sometimes even suggest obvious zoological nonsense. Since the problems involved in this phylogenetic conundrum encompass a wide array of analytical freedom and uncertainty it seems questionable whether a further increase in molecular data (quantity) can solve this classical deep phylogeny problem. This review thus strikes a blow for evaluating quality data (including morphological, molecule morphologies, gene arrangement, and gene loss versus gene gain data) in an appropriate manner. Ó 2012 Elsevier Inc. All rights reserved.
Contents 1. 2.
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Traditional morphological views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Modern molecular views . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Analyzing gene sequences (so-called ‘‘quantity data’’) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Analyzing gene presence and structure (so-called ‘‘quality data’’). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. Hox-/ParaHox genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Pax genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.3. Dicer genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.4. Leucine-rich repeat containing G protein-coupled receptors (LGRs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.5. Ribosomal genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.6. Mitochondrial genome characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary quantity versus quality data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. Traditional morphological views Three prominent urmetazoan hypotheses are most often under debate, (i) the placula, (ii) the planula and (iii) the gastrea hypothesis (Bütschli, 1884; Haeckel, 1874; Lankester, 1877, for overview see Kaestner, 1980). Common to all hypotheses is that the hypothetical ‘‘urmetazoon’’ must have had an extremely sim⇑ Corresponding author at: Division of Ecology and Evolution, Stiftung Tieraerztliche Hochschule Hannover, Bünteweg 17d, D-30559 Hannover, Germany. Fax: +49 511 953 8485. E-mail address:
[email protected] (B. Schierwater). 1055-7903/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ympev.2012.05.028
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ple morphology, perhaps just complex enough to pass the bridge between a protist and a metazoan. This bridge is the possession of more than one somatic cell type (protists may consist of dozens or hundreds of cells and may have more than one cell type within a colony but they never have two or more different somatic cells). Intrasomatic differentiation, which is the invention of the urmetazoon and a synapomorphy for all metazoans, became the motor for radiation of the metazoan bauplans (cf. Boero et al., 2007; Schierwater, B., de Jong, D., Desalle, R., 2009). Unfortunately for phylogenetics at the base of the tree, however, morphology was ‘‘frozen’’ as very subtle and uninterpretable anatomical changes occurred, and hence we are left with very
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few anatomical characters. In sharp contrast, the more derived root of the tree invented a third germ layer, the mesoderm, which fueled an explosion of bauplan radiation and new anatomical characters in the Bilateria. Placozoans meet the expectations of a primitive but general urmetazoan bauplan (see Fig. 1); they possess the simplest bauplan among extant metazoans (Grell and Benwitz, 1971; Grell and Ruthmann, 1991; Schierwater, 2005; Schulze, 1883, 1891). Only five somatic cell types (Guidi et al., 2011; Jakob et al., 2004) have been recognized. The simplicity of placozoans is further highlighted by their lack of any kind of axis of symmetry, organs, nerve and muscle cells, basal lamina, and an ultrastructurally identifiable extracellular matrix (note however, several cell–cell contact genes have been identified in Trichoplax; Chapman et al., 2010). In contrast to placozoans, Porifera usually possess more than a dozen somatic cell types (see Jiang and Xu, 2010; Valentine et al., 1994, for an overview of cell types in animals) and normally also develop an extracellular matrix (ECM) and basal membrane (e.g. Boury-Esnault et al., 2003) or even complete sealing epithelia (Adams et al., 2010). In Cnidaria and Ctenophora morphological complexity has increased. Thus many zoologists have been considering Placozoa as the closest living relative to the urmetazoon (e.g. Grell, 1971, 1982; Schulze, 1891). A larger group of researchers has seen the sponges, Porifera, closest to the base of the metazoan tree of life. The main argument here has been a single-character argument, the possession of the eye-catching character choanocytes, i.e. the morphological similarity between the choanocytes in the sponge gastrodermis and the single-celled choanoflagellates. But there also are other explanations for this observation than a non-simple evolutionary transformation of a choanoflagellate colony into a sponge bauplan. Some authors are in favor of a convergent evolution of collar structures and metazoan choanocytes (Maldonado, 2004) or even claim that the choanoflagellates are derived sponges. For example, several genes in the choanoflagellate genome (including genes for metazoan style cell adhesion and cell signaling; King et al., 2003; Manning et al., 2008) might be seen as indicators that evolution here went the opposite of the obvious way, i.e. that choanoflagellates originated from sponge
choanocytes, and not vice versa (Clark, 1868; Kent, 1878; Maldonado, 2004). In sum, there are currently two reasonable candidates for the closest living relative of the urmetazoon, Placozoa and Porifera.
2. Modern molecular views 2.1. Analyzing gene sequences (so-called ‘‘quantity data’’) Early molecular systematic studies used single gene data sets (mainly 18S or 28S rRNA) to resolve conflicts at the base of the metazoan tree of life. The resulting mixture of trees mostly supports the traditional view of early branching Porifera (for overview see Schierwater et al., 2010a), sometimes with the phylum Placozoa jumping inside a class of Cnidaria (Bridge et al., 1995; Siddall et al., 1995). Analysis of mitochondrial protein coding sequence data have promised to lead to a more reasonable picture of early animal evolution supporting a split between Bilateria and non-bilaterian animals with Placozoa branching first within the non-Bilateria (Dellaporta et al., 2006; Signorovitch et al., 2007). Nevertheless, analyses of recently sequenced mitochondrial genomes from Ctenophora (Kohn et al., 2012; Pett et al., 2011) highlight limitations of analyses based on mitochondrial protein sequence data. One of several problems relates to the observation that mitochondrial sequences in Ctenophora have evolved several times faster than in other animal phyla. The rapid progress in next generation sequencing techniques and computational data processing opens the door for phylogenetic analyses using large whole genome and EST data sets including several hundred or thousands of genes. As a consequence phylogenomic approaches to elucidate adaptive evolution in genes and genomes will become an important field in future research (cf. Goodman and Sterner, 2010; Shinzato et al., 2011). At present we are mostly limited to non-causal analyses of descriptive characters, like gene sequences. At the base of the metazoan ToL, the outcome of such analyses has been highly contradictory and can be summarized in three main scenarios:
Fig. 1. (A) Photograph of Trichoplax adhaerens, Schulze (1883). For additional images of placozoan specimens see www.trichoplax.com. (B) Modern placula hypothesis of metazoan origin (for details see Schierwater et al., 2009a). (from Schierwater et al., 2009a).
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Fig. 2. Deep metazoan relationships (A) The traditional view sees the Porifera in a basal position of the metazoan tree of life with Placozoa deriving next and Cnidaria and Ctenophora forming the Coelenterata. (B) The alternative traditional view sees Placozoa basal and in this case the Porifera deriving next. (C) A modified view of the traditional scenario in (B) supports a sister group relationship between Bilateria and non-bilaterian animals with Placozoa branching first within the non-Bilateria. (D) One of the surprising topologies that derive from certain molecular analyses which put the morphologically derived Ctenophora in a basal position.
(i) The traditional ‘‘Porifera first scenario’’ is supported by some recent molecular studies by, e.g. Philippe et al. (2009), Fig. 2A). Despite of the large data sets and the optimized phylogenetic analyses several important nodes and especially the position of Placozoa are not well supported in these phylogenetic inferences and some studies even doubt the monophyly of sponges (e.g. Sperling et al., 2009). However, recent studies, e.g. on the evolution of post-synaptic proteins (Alie and Manuel, 2010) use a ‘‘Placozoa basal’’ scenario (Fig. 2B) to reconstruct the most parsimonious way of evolution of these proteins in Metazoa. (ii) A second scenario has been suggested in a study combining morphological and molecular data (from both nuclear and mitochondrial genes) in one concatenated data matrix (Schierwater et al., 2009a, Fig. 2C). This study postulates the monophyly of the Porifera but places them in a more derived position within the diploblasts with Placozoa as the earliest branching diploblast phylum and Cnidaria and Ctenophora forming a ‘‘Coelenterata’’ clade (Leuckart, 1848). The most remarkable point, however, is the sister group relationship of the monophyletic Bilateria and the monophyletic non-bilaterians (i.e. Placozoa, Porifera, Cnidaria and Ctenophora) with high support values. A subsequent study with increased taxon sampling (Schierwater et al., 2009b) as well as mitochondrial sequence data (Dellaporta et al., 2006) gave further support for this scenario. (iii) A third scenario with the morphologically complex Ctenophora as the earliest branching metazoan phylum has been suggested based on molecular data only (Dunn et al., 2008; Hejnol et al., 2009, Fig. 2D). With special regard to the base of the metazoan tree of life the latter, hot debated scenario not only suggested Ctenophora as sister group to all other animals but also placed Placozoa between the Demospongiae and Homoscleromorpha supporting paraphyly of sponges. A most recent analysis shows that the positioning of the highly derived Ctenophora is likely the result of a long branch attraction (LBA) artifact (Nosenko et al., unpublished data). Several studies (e.g. Philippe et al., 2011; Pick et al., 2010) have re-analyzed the previously mentioned data sets and list several key factors, which strongly influence the outcome of the analyses. It has been shown that the choice of outgroups, the ingroup taxon
sampling, misidentified taxa, the inclusion of non-orthologous or saturated genes, chimeric sequences or an inappropriate evolutionary model can lead to long branch attraction artifacts and well supported clades which are in conflict with zoological knowledge (Philippe et al., 2011). 2.2. Analyzing gene presence and structure (so-called ‘‘quality data’’) There are several approaches to infer evolutionary relationships based on the presence or absence or genomic organization of genes of a distinct gene family. We just list a few examples. 2.2.1. Hox-/ParaHox genes Hox-/ParaHox genes play important roles in pattern formation along the anterior-posterior axis in bilaterian animals (Carroll, 1995). Both the inventory and the structure of Hox-/ParaHox-like genes suggest an early diploblast-bilateria split and provides evidence for the placozoan Trox-2 gene being the Proto-Hox/Parahox gene for all metazoans (Jakob et al., 2004). Since sponges and ctenophores probably do not possess any Hox genes, the above scenario assumes a loss of Hox-/ParaHox-like genes in both phyla (for literature and controversial views see Chourrout et al., 2006; Garcia-Fernandez, 2005; Ryan et al., 2007; Schierwater and Kamm, 2010). Based on the presence of Hox/Parahox genes the so-called taxon ‘‘ParaHoxozoa’’ was suggested, which includes Placozoa, Cnidaria and Bilateria (Ryan et al., 2010) and unifies two distantly related animal phyla, the Porifera and Ctenophora, as a sister group to this so-called ‘‘subkingdom’’. If ‘‘ParaHoxozoa’’ are meant as a natural rather than an artificial clade, this scenario must be seen with great caution, however. Besides the zoological problem of uniting two distinct phyla without any clade diagnostic morphological or developmental synapomorphy, the loss of a single gene (Proto Hox/ParaHox gene) is not a very strong character. If one wants to assume that neither Porifera nor Ctenophora ever possessed a Hox like gene and that they form a monophyletic group, this would put a highly derived animal group (Ctenophora) together with one of the basal groups (Porifera) and would create sharp conflicts to our knowledge from comparative zoology. Some people may also argue that gene information from a yet incomplete genome of a single species may not necessarily be representative for the phylum. Thus, the question whether Hox-like genes are indeed absent in Porifera and Ctenophora remains open. If absent, the question whether this is an ancestral or derived feature
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Fig. 3. Pax gene evolution model. Based on the structural features of Pax genes, i.e. paired domain(s), homeodomain and octapeptide, an evolutionary scenario has been suggested in which a PaxB-like Ur-Pax gene is the starting point for several duplication events that led to a large diversity of Pax gene subfamilies in Cnidaria and Bilateria (for details see Hadrys et al., 2005). Paired domains (PD) = green rectangles, homeodomains (HD) = black rectangles, octapeptides (OP) = blue circles. (from Hadrys et al., 2005). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
remains open, too. According to current knowledge placozoans possess the most ancient Hox-/ParaHox system in the Metazoa. 2.2.2. Pax genes The Pax gene family consists of tissue specific transcription factors that are important in early animal development for the specification of tissues and organs (Chi and Epstein, 2002). The structure of Pax genes (i.e. the partial or complete presence of homeo- and paired domains and octapeptide motifs) suggests that a putative Proto-Pax gene was similar to the PaxB gene found in Trichoplax (Hadrys et al., 2005; Fig. 3). Sponges also possess a single PaxB gene. Pax genes from Ctenophora have not been described yet. The Cnidaria and Bilateria show a large radiation of Pax genes (Hill et al., 2010). Thus, the sum of available evidence is congruent with the hypothesis that the placozoan Pax gene is ancestral to the Pax genes in other phyla, but the data set may be incomplete yet. 2.2.3. Dicer genes Proteins of this gene family are essential components of the RNA interference pathway (for review see McManus, 2004). A recent study (de Jong et al., 2009) has shown that the Placozoa is the only known metazoan phylum which contains both representatives of an ancient duplication event. Like all other metazoan animals, sponges only possess group II dicer proteins. These data are consistent with the hypothesis that only the Placozoa still harbor the direct descendents of a ‘Proto-Dicer’ gene. 2.2.4. Leucine-rich repeat containing G protein-coupled receptors (LGRs) LGRs are seven-transmembrane domain receptors with important functions in development and reproduction. They include such prominent proteins as the relaxin receptors in mammals, as well as
the bursicon receptor in insects (cuticle hardening) (Van Loy et al., 2008). Based on phylogenetic inferences and structural data it has been proposed that placozoan LGRs may represent an ancient form of metazoan LGRs (Van Hiel et al., 2011). Comparative data from sponges and ctenophores are missing yet. 2.2.5. Ribosomal genes Ribosomal genes show a tendency for size reduction from protists to higher metazoans. An illustrative example is shown in Ender and Schierwater (2003), where placozoans possess the longest and most complex 16S rRNA morphology. Both size and complexity get gradually reduced from Placozoa to Porifera to Cnidaria and finally Bilateria. Ctenophores show by far the most reduced 16S rRNA genes of all basal metazoans (Pett et al., 2011; Kohn et al., 2012). Again, The parsimonious interpretation of the observed molecule morphology data is that placozoans mirror the most ancestral condition within extant metazoans. 2.2.6. Mitochondrial genome characteristics With respect to gene presence/absence and gene arrangement data mitochondrial (mt) genomes provide a rich repertoire of phylogenetically informative characters at different taxonomic levels. It is believed that one of the strongest characters for phylogenetic reconstruction is gene loss. The latter occurs regularly, but the reverse, i.e. regaining and incorporating a formerly lost gene into a mitochondrial genome, must be an extremely rare event. If several genes need to be regained and incorporated the likelihood for such an event jumps beyond probability values generally accepted in biology. The placozoan mt genomes represent the most complete mt genomes among metazoans and according to the phylogenetic interpretation of gene loss data the placozoan mitochondrial genomes would represent the most ancestral condition found in
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Fig. 4. Overall tendencies in early metazoan mitochondrial genome evolution. Mitochondrial genome evolution from Protozoa (represented by Choanoflagellata) to early metazoan phyla (Placozoa, Porifera, Cnidaria and Ctenophora). A clear tendency to genome compaction is visible (see text). A special case of mt genome evolution has been described in Cnidaria, where ancestrally circular chromosomes become linearized or even linear fragmented in more derived cnidarian taxa.
metazoans. For example, the number of open reading frames (ORFs) in Placozoa is up to eight (Dellaporta et al., 2006; Signorovitch et al., 2007), while sponges and cnidarians only exceptionally harbor up to three. In metazoan outgroups the number of ORFs is usually high, e.g. in the choanoflagellate Monosiga brevicollis we find 6 additional ORFs while the ichthyosporean Amoebidium parasiticum harbors at least 24 (Burger et al., 2003). Obviously a high number of ORFs is an ancestral metazoan feature. From protozoans to derived metazoans mitochondrial genomes show a clear tendency for the reduction of non-coding intergenic spacer regions and genome size in general (Fig. 4). In protist groups such as Ichthyosporea, Filasterea and Choanoflagellata large mitochondrial genomes around 60 kb or more are the normal case. As several molecular studies support a sister group relationship between the Choanoflagellata and metazoans the large mt genome of Monosiga brevicollis (76 kb) might be seen as a reference mt genome for early metazoans. The largest animal mitochondrial genomes have been found in placozoans (32–43 kb), the second largest mt genomes in sponges (16–29 kb), and cum grano salis normal sized mitochondrial genomes in almost all other metazoans (16–18 kb). An interesting apomorphy is seen in Cnidaria, where the basal Anthozoa harbor circular mt genomes of 15–21 kb size whereas the derived Scyphozoa, Hydrozoa, Staurozoa and Cubozoa have linearized and sometimes highly fragmented mitochondrial genomes (up to eight chromosomes in cubozoans) with a total length of up to 28 kb in cubozoans (Smith et al., 2012, for overview see Kayal et al., 2012). The most derived mt genomes are found in the Ctenophora, where the overall mt genome size is only about 11 kb and the number of encoded genes as well as the coding sequence of identified genes is highly reduced (Kohn et al., 2012; Pett et al., 2011). In addition to the presence of additional open reading frames and large intergenic regions in the placozoan mitochondrial genomes, these also harbor several introns as well as a full complement of 24 tRNA genes. Although introns are regularly also found in Porifera and Cnidaria the trans-splicing group I introns in Placozoa seem to be unique to this phylum (Burger et al., 2009). In higher animal phyla introns or intergenic spacers are rare or absent features. In Bilateria, but also already seen in Porifera, Cnidaria and Ctenophora, the reduction has progressed to a degree that genes are overlapping. With respect to mitochondrial encoded
tRNAs especially Cnidaria or Ctenophora tend to reduce the number of encoded tRNA genes within non-bilaterian animals down to one tRNA. In Porifera the number of tRNA ranges from some 2 to 27, mirroring both secondary reduction and duplication events. Summarizing all of the mitochondrial jigsaw pieces the placozoan mt genome shows the highest similarity to protozoan mt genomes and almost looks like an evolutionary link between protists and metazoan mt genomes. Whether the unequivocal basal mt genome features of the Placozoa also mirror a basal position for the phylum may be seen as a different question. 3. Summary quantity versus quality data The sum of molecular trees based on large numbers of gene sequences does not resolve phylogenetic relationships at the base of the Metazoa. Conflicting scenarios have been published in short sequence and each single analysis can be criticized for one or the other reason. It is unclear to many whether the base of Metazoa can ever be resolved by means of sequence data even if whole genomes and extensive taxon sampling is used. The opposite data type, i.e. the so-called ‘‘quality data’’ in the form of anatomical, developmental, molecule morphology, gene loss, gene structure and gene arrangement data is comparatively very limited with respect to the number of available characters, but provides less confusion. The majority of the data support a basal position of the Placozoa near the root of the metazoan tree of life. The real problem, however, remains completely unresolved, the problem of the relative character weighting between ‘‘quality data’’ on the one hand and molecular sequence data on the other hand. Solving this problem is far away from trivial but it must eventually be addressed in order to be able to perform concatenated analyses in a useful way, particularly at the most difficult part of the metazoan tree of life, its root. Acknowledgments We thank Rob DeSalle (AMNH, New York) and Stephen Dellaporta (Yale University) for intellectual input and comments on the manuscript. H.J.O acknowledges a doctoral fellowship from the Studienstiftung des deutschen Volkes. M.E. acknowledges
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funding by the Evangelisches Studienwerk e.V. Villigst, the Stiftung Tierärztliche Hochschule Hannover, the German Academic Exchange service (DAAD) and the support by the Swire Institute of Marine Science. Our work has been funded by the German Science Foundation (DFG Schi 277/20-3 and Schi 277/26-1). Special thanks to Max. References Adams, E.D.M., Goss, G.G., Leys, S.P., 2010. Freshwater sponges have functional, sealing epithelia with high transepithelial resistance and negative transepithelial potential. Plos One 5. Alie, A., Manuel, M., 2010. The backbone of the post-synaptic density originated in a unicellular ancestor of choanoflagellates and metazoans. BMC Evolution. Biol. 10, 34. Boero, F., Schierwater, B., Piraino, S., 2007. Cnidarian milestones in metazoan evolution. Integrat. Comp. Biol. 47, 693–700. Boury-Esnault, N., Ereskovsky, A., Bezac, C., Tokina, D., 2003. Larval development in the Homoscleromorpha (Porifera, Demospongiae). Invertebr. Biol. 122, 187– 202. 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