Phylogenetic-Signal Dissection of Nuclear Housekeeping Genes Supports the Paraphyly of Sponges and the Monophyly of Eumetazoa

“Eumetazoan Monophyly Several recent studies using data sets such as ESTs (Dunn et al. 2008; Schierwater et al. 2009) and complete mitochondrial genomes (Lavrov et al. 2008) have found overall topologies for the most basal part of the metazoan tree that are radically different from the one found in…”

“It is worth mentioning that the monophyly of Porifera is not unequivocally accepted [28,46]; only the analysis of 30,000 positions with a rich taxon sampling and a complex model of evolution recovers it with significant statistical support [3].”

“’More recent studies alternatively support paraphyly [63] or holophyly [58, 64] of sponges.”

“Whether sponges comprise a monophyletic sister group to all other animals (Figure 3) or a paraphyletic group from which other animals emerged is still debated (Phillipe et al. 2009, Sperling et al. 2009).”

“Some studies suggested that Porifera is paraphyletic (Sperling et al. 2009), but these studies considered a relatively small number of nuclear genes.”

“We store the information about bipartition merging behavior in a data structure called merger graph .”

“To further test this idea, we reinvestigated another difficult question pertaining to deep animal relationships (Dunn et al. 2008; Hejnol et al. 2009; Philippe et al. 2009; Sperling et al. 2009; Pick et al. 2010; Philippe, Brinkmann, Lavrov, et al. 2011) by introducing missing data in the 150 genes of Pick et al.”

“) Taxa Genes Sites Gene occupancy, % Missing data (including gaps), % Full dataset (1) 76 251 81,008 75 40 Certain paralogs removed (2) 76 250 80,735 75 40 Taxa with high LB scores removed (3) 70 250 80,735 75 41 Genes with high LB scores removed (4) 70 231 73,323 75 41 All outgroups except Choanoflagellates removed (…”

“) [24,33].”

“Based on our previously described phylostratigraphic procedure [1,2], we generated a database of genome sequences divided into 19 phylostrata corresponding to the evolutionary relationships of the major taxa supported by phylogenomic analyses (Figure 1) [15-19].”

“…groups is monophyletic; the only exception in recent literature has been the proposition that Porifera is paraphyletic, giving rise to all other animals (Sperling et al. 2009a, 2010), but this has been contradicted by much larger samples of genes that defend sponge monophyly (Philippe et al. 2009).”

“There has been debate concerning the monophyly versus paraphyly of sponges and whether ctenophores are a sister clade to sponges plus Eumetazoa, or whether ctenophores are themselves eumetazoans (Sperling et al., 2009; Nosenko et al., 2013).”

“Although attempts to use such markers, for example microRNAs to resolve sponge relationships (Sperling et al., 2010), transposable elements (short interspersed elements, SINEs; Piskurek and Jackson, 2011) and changes in spliceosomal intron positions (NIPs; Lehmann et al., 2012), to resolve early…”

“We next performed a signal-dissection analysis (13, 28), based on the slow-fast technique (27).”

“…the last common ancestor of animals was benthic, sessile, and fed using the WCS (Cavalier-Smith et al. 1996; Collins 1998; Kruse et al. 1998; Borchiellini et al. 2001; Medina et al. 2001; Peterson and Eernisse 2001; Wallberg et al. 2004; Peterson and Butterfield 2005; Sperling et al. 2007, 2009).”

“Consistently with our results, some of themost thorough analyses to date (20, 21) agree that the Placozoa are the sister group of Neuralia, even though some investigators (22, 30) found different results.”

“This clade has been named “Democlavia” by Sperling et al. (2009); however, this name 154 P. Cárdenas et al. is a nomen nudum because it has not been given a formal Linnaean (rank-based) definition.”

“When hexactinellids are excluded and only demosponges analyzed (whose branch lengths for this data set are similar to those of eumetazoans; Sperling et al., 2009), a spicule gap of approximately 140 Myr still remains between the node Haplosclerida + Democlavia and the first appearance of spicules…”

“2013), whereas others have suggested that Myxospongiae may be the sister group to all other demosponges (Sperling et al. 2009; Bayesian analyses of Hill et al. 2013; Thacker et al. 2013).”

“vectensis is considered to be the closest outgroup to the bilaterians among the known extant taxa (Finnerty et al. 2004; Dunn et al. 2008; Hejnol et al. 2009; Sperling et al. 2009; Ryan et al. 2010).”

“In contrast, marine and freshwater Haplosclerida form a well-supported clade in phylogenetic reconstructions based on nuclear housekeeping genes (Sperling et al., 2009).”

“Whether sponges are monophyletic (Philippe et al., 2009) or paraphyletic (Sperling et al., 2009), they are certainly the sister group or grade of all other animals (Philippe et al., 2011).”

“Evolutionary relationships across the most diverse class of Porifera, Demospongiae, have mainly been addressed with three sets of phylogenetic markers: ribosomal DNA sequences [17,42], complete mitochondrial genome sequences [43], and amino acid sequences that code for seven nuclear housekeeping genes [18,44].”

“This is consistent with recent large-scaled molecular results confirming that molluscs are non-platyzoan lophotrochozoans (Dunn et al. 2008, Sperling et al. 2009, Pick et al. 2010).”

“[7-10], some recent phylogenomic analyses place ctenophores more basally [11,12] calling into question our understanding of the evolution of nerves and the ancestral metazoan state.”

“Failure to detect paralogues can lead to misinterpretations of cellular biochemistry, and often inaccuracies in reconstructions of phylogeny and molecular evolution [71,72].”

“Should the alternative hypothesis that Porifera is paraphyletic (Sperling et al., 2009) gain significant support in the future and the sponge monophyly hypothesis be convincingly refuted, Ho-moscleromorpha and Calcarea would need to change ranks and be reconsidered as potential distinct phyla.”

“The phylogenetic relationships of extant sponges are contentious; recent molecular phylogenetic studies consistently resolve them as sister to all other animals, but whether they are monophyletic or paraphyletic is under debate (Philippe et al., 2009; Sperling et al., 2009).”

“Recent work has shown that the latter group, Homoscleromorpha, has a BM structure containing type IV collagen (Boute et al., '96), which some say is evidence of a “true epithelium” and which has prompted use of the term “Epitheliozoa” (Boury‐Esnault et al., 2003; Sperling et al., 2009).”

“These approaches, along with resolving the exact order in which these early-branching metazoans diverged from the lineage leading to bilaterians (that is, whether sponges or ctenophores are the sister group to all other animals and whether sponges are monophyletic [34,38]), provide an opportunity to reconstruct the ev…”

“However, as recent molecular phylogenomic and phylogenetic analyses both support [1,15] and reject [3,4,16,17] this traditional view, it remains unclear as to whether sponges or ctenophores are the sister group to all other animals and whether poriferans are monophyletic.”

“If these sponges are paraphyletic (e.g., Sperling et al. 2009), then these may represent characters that evolved in the clade containing the Calcarea, Homoscleromorpha, and Eumetazoa after it diverged from the lineage comprising the majority of demosponges.”

“Democlavia [1,2].”

“Among these recent phylogenetic studies based on nuclear sequences, two alternative positions of homoscleromorphs have emerged: (i) closer to eumetazoan than to the other sponges, resulting in the paraphyly of sponges [7,9,11,23,24] or (ii) as the sister group of calcareous sponges within monophyletic Porifera [8,12].”

“Porifera is considered one of the most ancient extant animal phyla (Borchiellini et al. 2001; Medina et al. 2001; Cavalier-Smith and Chao 2003; Philippe et al. 2009; Sperling et al. 2009; but see Dunn et al. 2008; Schierwater et al. 2009) and thus represents a critical comparative model to gain a general”

“The non-monophyly of the Porifera (as seen in all of our analyses) has been observed in several other molecular studies based on nuclear genes, where at least the Homoscleromorpha do not group with other sponges (e.g. Hejnol et al., 2009; Sperling et al., 2009; Nosenko et al., 2013).”

“Porifera encompass four distinct taxonomic classes (Philippe et al. 2009; Sperling et al. 2009; Erwin et al. 2011) with differences in junction depending on the class.”

“Porifera, the sponges, are the oldest branching group of animals (Philippe et al. 2009).”

“site stripping) from the analyses (e.g. Brinkmann and Philippe, 1999; Rodriguez-Ezpeleta et al., 2007; Pisani, 2004; Sperling et al., 2009; Rota-Stabellli et al., 2010).”

“The most current phylogenomic analyses of animal relationships place sponges as most basal [1,5,8], but other”

“Less well appreciated is the observation that the sorting of sites based on character state variation or compatibility criteria allows the properties of sites that impact on tree building to be more easily studied (Sperling et al. 2009).”

“A number of multi-gene and phylogenomic analyses that did not include Ctenophora found a sistergroup relationship of Cnidaria to Bilateria (Srivastava et al. 2008, 2010; Sperling et al. 2009; Erwin et al. 2011).”

“Because of the extreme rate variation and presence of homoplasy-prone sites, this dataset is expected to be difficult to analyze and likely to support phylogenetic artifacts even when analyzed using well-fitting, parameter-rich models (see Sperling et al. 2009a).”

“Finally, before systematics entered the high-throughput era, a handful of works used concatenated alignments (from 7 to 23 genes) for a significant number of phyla (Bourlat et al., 2008; Paps et al., 2009a; Sperling et al., 2009).”

“Such files, for instance, (*.plot) are automatically produced by the COMPASS program by Simon Harris (www.ncl.ac.uk/ microbial_eukaryotes/downloads.html) in which compatibility-based sorting approach is implemented (Pisani 2004; Sperling et al. 2009).”

“, [1,2,3,4,5,6,7,8,9]) towards the mining of genomic and transcriptomic data using hundreds of genes (e.”

“…and 28S rRNA (Borchiellini et al. 2004), 28S rRNA and CO1 (Nichols 2005), 28S rRNA (Holmes and Blanch 2007), complete mitochondrial genomes (Lavrov et al. 2008), and housekeeping genes (Sperling et al. 2009; Hill et al. 2013) are congruent and show Haploscleromorpha as sister to Heteroscleromorpha.”

“The tree topology represents a synthesis of phylogenetic reconstructions from [66,74–75], showing the twelve sponge species from eight different sponge families targeted in this study.”

“This hypothesis predicts Echinodermata Malacostraca Diptera Metatheria Primates Laurasiatheria Crocodylidae Aves Squamata Serpentes Caudata Actinopterygii Anura Porifera Cnidaria 0.1 1.0 1.0 0.96 0.97 1.0 0.83 0.62 1.0 0.76 0.94 0.94 Bi lat er ia Ve rte br ata Mammalia Sauria Amphibia Pancrustacea Figure 3 Topology in…”

“However, analyses performed with only characters carrying a moderate evolutionary rate or with the CAT + mtArt, which has been confirmed as an effective model to overcome the effects of Long Branch Attraction (LBA) [68], consistently resolved a monophyletic Branchiopoda clade.”

“data is consistent with a monophyletic Haplosclerida [14].”

“Among the non-bilaterian animals, recent utilization of phylogenomics has increased our understanding of metazoan relationships [1-5].”

“…with the fact that basal lamina–like structures have not been observed in demosponges via electron microscopy (Leys et al. 2009), with the exception of homoscleromorph sponges, which are now thought to represent a lineage distinct from other demosponges (Philippe et al. 2009; Sperling et al. 2009).”

“Although accommodating ASRV is always recommended, how to best model this variation is an area of active research (Sperling et al. 2009; Cummins &McInerney 2011; Stabelli et al. 2013; Rajan 2013).”

“Hypothesis testing with rRNA data rejects the traditional, morphology-based idea that the homoscleromorph sponge, Oscarella, is a demosponge (Table 4, Hypothesis 15), and developmental evidence now upholds this rejection (Boury-Esnault et al., 2003; Kober and Nichols, 2007; Sperling et al., 2009a).”

“There is an ongoing debate on the molecular phylogeny aspects of basal metazoans (Dohrmann et al. 2008; Srivastava et al. 2008; Philippe et al. 2009; Sperling et al. 2009), and although we did not address this issue directly, we hope that the data provided in this paper will provide further evidence for understanding…”

“Note that this interpretation is independent of whether sponges form a monophyletic or paraphyletic group (Collins 2002; Jackson et al. 2007; Sperling et al. 2009; Erwin et al. 2011) but places Placozoa as the outmost group within all other Metazoa, and sponges as an outgroup to this common clade…”

“Sponge paraphyly has commonly been reported when using ribosomal (e.g. Borchiellini et al. 2001, 2004), nuclear housekeeping (e.g. Sperling et al. 2009) and mitochondrial markers (e.g. Lavrov et al. 2008).”

“Although lack of unambiguous morphological support for Ecdysozoa persists as a moot point, in the light of overwhelming molecular evidence and lack of morphological evidence conclusively discrediting Ecdysozoa, is it now finally time to shed the notion of Coelomata?”

“This concept called Neotrochozoa is supported by some molecular studies, e.g. using housekeeping genes (Peterson et al. 2008, 2009; Sperling et al. 2009) or EST data (Kocot et al. 2011) with, however, still limited taxon sampling.”

“Differences in taxon sampling appear to have been a major reason for these divergent results (Sperling et al. 2009a).”

“…approaches, such as EST projects (e.g., Dunn et al., 2008; Philippe et al., 2009; Pick et al., 2010); (2) the sequencing of specific genomic DNA markers (e.g., Borchiellini et al., 1998; Erpenbeck et al., 2005b); and (3) the sequencing of specific cDNA markers (Sperling et al., 2007, 2009).”

“The latter result contradicts the current view that sponges are either monophyletic [25] or that homoscleromorphs are closer to cnidarians [26].”

“Nuclear protein-coding genes have been employed rarely in sponges (Sperling et al. 2009; Hill et al. 2013; DeBiasse et al. 2014), but given their fast rates (relative to COI) and the increasing ease of next generation data collection, nuclear loci hold promise for future phylogeographic and phylogenetic studies in spo…”

“Recent studies using molecular phylogenetic analyses, transcriptomic and genomic data either confirm [14–16] or reject [17–20] the view of sponges as the sister group to all other animals.”

“The comparison of phylogenies obtained using differently fitting substitution models and different partitions has previously been used to explore the influence of non-phylogenetic signal on phylogenetic analyses [27, 29, 41–43].”

“Ctenophora possesses sequences that share a common ancestor with the bilaterian ciliary opsins, and the position of the ciliary opsins in the minimal opsin tree suggests that the shared ancestor of Ctenophora, Cnidaria, and Bilateria possessed three opsins.”