1 Current understanding of Ecdysozoa and its internal phylogenetic 2 relationships 3 4 Gonzalo Giribet1 and Gregory D. Edgecombe2 5 1 Museum of Comparative Zoology, Department of Organismic and Evolutionary Biology, 6 Harvard University, 26 Oxford Street, Cambridge, MA 02138, USA, ggiribet@g.harvard.edu 7 2Department of Earth Sciences, The Natural History Museum, Cromwell Road, London SW7 8 5BD, UK, g.edgecombe@nhm.ac.uk 9 10 11 Synopsis Twenty years after its proposal, the monophyly of molting protostomes— 12 Ecdysozoa—is a well-corroborated hypothesis, but the interrelationships of its major 13 subclades are more ambiguous than is commonly appreciated. Morphological and molecular 14 support for arthropods, onychophorans and tardigrades as a clade (Panarthropoda) 15 continues to be challenged by a grouping of tardigrades with Nematoida in some molecular 16 analyses, although onychophorans are consistently recovered as the sister group of 17 arthropods. The status of Cycloneuralia and Scalidophora, each proposed by morphologists 18 in the 1990s and widely employed in textbooks, is in flux: Cycloneuralia is typically non- 19 monophyletic in molecular analyses, and Scalidophora is either contradicted or 20 incompletely tested because of limited genomic and transcriptomic data for Loricifera, 21 Kinorhyncha and Priapulida. However, novel genomic data across Ecdysozoa should soon 22 be available to tackle these difficult phylogenetic questions. The Cambrian fossil record 23 indicates crown-group members of various ecdysozoan phyla as well as stem-group taxa 24 that assist with reconstructing the most recent common ancestor of panarthropods and 25 cycloneuralians. 1 26 A history of Ecdysozoa 27 Few studies have revolutionized the field of animal systematics as much as the phylogenetic 28 analysis of 18S rRNA sequence data of a handful of metazoans published by Aguinaldo et al. 29 (1997) in which they proposed the clade Ecdysozoa, a monophyletic group of animals that 30 molt their cuticle during the life cycle. The original analysis included members of the 31 ecdysozoan phyla Arthropoda 1, Onychophora, Tardigrada, Nematoda, Nematomorpha, 32 Kinorhyncha and Priapulida (molecular data for Loricifera were unavailable at the time). 33 This study was soon followed by other molecular and morphological analyses corroborating 34 or discussing the relevance of Ecdysozoa (e.g., Giribet 1997; Giribet and Ribera 1998; 35 Schmidt-Rhaesa et al. 1998). Ecdysozoa, as understood nowadays (see different 36 configurations in Fig. 1), includes the members of three putative subclades, Nematoida 37 (composed of Nematoda and Nematomorpha), Scalidophora (Priapulida, Loricifera and 38 Kinorhyncha) and Panarthropoda, the latter being ecdysozoans with paired ventrolateral 39 segmental appendages, i.e., Arthropoda, Onychophora and Tardigrada (Fig. 1a). The first 40 two are commonly grouped together as Cycloneuralia (Fig. 1a), the name referring to a ring41 shaped circumpharyngeal brain. 42 Since these early analyses, Ecdysozoa has been supported by a diverse source of 43 data, both morphological and molecular (but see Wägele et al. 1999; Wägele and Misof 44 2001; Pilato et al. 2005), contradicting the longstanding hypothesis of panarthropods being 45 closely allied to annelids in the clade Articulata (Haeckel 1866), for which segmentation was 46 the unifying character. Some authors tried to reconcile the Articulata and Ecdysozoa 47 hypotheses by providing intermediate evolutionary scenarios between these two groups 48 (Nielsen 2003), but no data have supported this scenario. Molecular analyses occasionally 49 fell victim to common biases, and placed additional taxa within Ecdysozoa, notably the 1 Euarthropoda sensu Ortega-Hernández (2016); see that paper for a historical account of the use of names such as Arthropoda, Euarthropoda, Tactopoda and others. 2 50 unstable Chaetognatha (e.g., Zrzavý et al. 1998; Paps et al. 2009), now thought to be related 51 to Gnathifera (Frröbius and Funch 2017), and Buddenbrockia (Zrzavý et al. 1998), since 52 reassigned with confidence to Myxozoa (Jiménez-Guri et al. 2007). Likewise, early 53 phylogenomic analyses restricted to a handful of available genomes proposed non54 monophyly of Ecdysozoa, often favoring a group called Coelomata that united arthropods 55 with chordates to the exclusion of nematodes (Wolf et al. 2004; Philip et al. 2005; Rogozin et 56 al. 2007), but that hypothesis was soon refuted with improved evolutionary models 57 (Lartillot et al. 2007). Virtually all subsequent phylogenomic analyses have found support 58 for Ecdysozoa (e.g., Philippe et al. 2005; Irimia et al. 2007; Dunn et al. 2008; Hejnol et al. 59 2009). That is not however the case from mitogenomics (Podsiadlowski et al. 2008; Rota60 Stabelli et al. 2010; Popova et al. 2016), but as of today, no mitochondrial genomes are 61 available for Nematomorpha or Loricifera—and some loriciferans may altogether lack 62 mitochondria (Danovaro et al. 2010; Danovaro et al. 2016). 63 Although Ecdysozoa was originally portrayed by some to be an artifact of flaws in 64 molecular systematics (Wägele and Misof 2001), morphologists had already implicitly or 65 explicitly questioned Articulata while instead supporting a clade that unites molting 66 protostomes. Eernisse et al. (1992) published a phylogenetic analysis of a morphological 67 data matrix resolving Panarthropoda with Nematoda and Kinorhyncha (Priapulida was left 68 unresolved in a basal protostome trichotomy), while recognizing the annelid lineages as 69 part of Spiralia. This visionary phylogeny of bilaterians received little subsequent attention, 70 but clearly spoke in favor of morphological arguments that conflict with Articulata. Even 71 before Eernisse et al. (1992), Crowe et al. (1970) mentioned the similarity in the 72 organization of the cuticles of tardigrades and nematodes and how “On this basis a 73 phylogenetic affinity of tardigrades for nematodes was supported”. Later, while discussing the 74 phylogenetic position of the recently discovered loriciferan body plan (Kristensen 1983), R. 3 75 M. Kristensen stated that “Annulation of the flexible buccal tube, telescopic mouth cone, and 76 the three rows of placoids are found only in Tardigrada and Loricifera (Kristensen, 1987). 77 Because tardigrades exhibit several arthropod characters (see Kristensen, 1976, 1978, 1981), 78 this last finding supports a theory about a relationship between some aschelminth groups and 79 arthropods (Higgins, 1961). That theory has recently gained support derived primarily from 80 new ultrastructural data, e.g., the fine structure of the chitinous cuticular layer, molting cycle, 81 sense organs, and muscle attachments.” (Kristensen 1991: p. 352). This hypothesis had 82 already been postulated in Higgins’ unpublished PhD thesis, and morphological support for 83 Ecydysozoa and/or inconsistencies with Articulata were proposed soon after the 84 publication of the seminal molecular paper by Aguinaldo et al. (e.g., Kristensen 2003; Giribet 85 2004; Mayer 2006; Koch et al. 2014). A key implication of the acceptance of Ecdysozoa is 86 thus whether the annelid and panarthropod segmentation is homologous, and if so, at what 87 level (see discussions in Scholtz 2002; Giribet 2003; Scholtz 2003; Minelli 2017). 88 89 Is there morphological support for Ecdysozoa? 90 Several authors tried to articulate a few morphological characters that could be apomorphic 91 for Ecdysozoa, most related to their cuticle—cuticles are present across the animal kingdom 92 but are difficult to define (Rieger 1984; Ruppert 1991). Some of the proposed cuticular 93 characters include its trilayered ultrastructure and the formation of the epicuticle from the 94 tips of epidermal microvilli, annulation, molting (probably through ecdysteroid-mediated 95 hormones), or lack of cilia for locomotion (Schmidt-Rhaesa et al. 1998). Other characters 96 include the terminal position of the mouth (Giribet 2003), a character that like annulation, 97 is often found in Cambrian ecdysozoan fossils that have been assigned to the stem groups of 98 lineages whose extant members have secondarily modified this trait. Recent developmental 99 data, however suggest that the terminal mouth of priapulans has a ventral embryological 4 100 origin, which the authors interpret as the ancestral state in ecdysozoans (Martín-Durán and 101 Hejnol 2015). 102 Some of these characters, especially the annulated cuticle and the terminal mouth 103 are prevalent in many Cambrian fossils, including stem-group arthropods such as 104 Kerygmachela (see Budd 1998), possible stem-group onychophorans such as Collinsium (see 105 Yang et al. 2015), lobopodians of uncertain systematic position such as Onychodictyon (Ou 106 et al. 2012), and lobopodians that are either allied to tardigrades or near the base of 107 Panarthropoda such as Aysheaia (Fig 2D). The annulated cuticle, however, does not occur in 108 many modern ecdysozoans (it is only present in Priapulida, Onychophora and some 109 Nematoda), and the mouth has a ventral position in some Tardigrada, in Onychophora, and 110 in most Arthropoda. While segmentation exists in four of the seven ecdysozoan phyla, it is 111 unclear how many times it evolved, and at least it would have originated independently in 112 Kinorhyncha and Panarthropoda—but the unstable position of Tardigrada makes this 113 inference difficult. 114 One of the characteristics often cited for Ecdysozoa is the presence of α-chitin in 115 their cuticle, but to date this has only been found in Priapulida and Panarthropoda (Greven 116 et al. 2016). In addition, the cuticle of Pentastomida, which are bona fide members of the 117 crustacean–hexapod clade, Tetraconata or Pancrustacea (Abele et al. 1989; Giribet et al. 118 2005; Regier et al. 2010; Oakley et al. 2013; Rota-Stabelli et al. 2013; Li et al. 2016), contains 119 ß-chitin (Karuppaswamy 1977). No information is yet available about the type of chitin 120 present in the other members of Ecdysozoa. 121 The evolution of the ecdysozoan nervous systems have centered on understanding 122 the nature of the brain, which is circumoral in the non-panarthropods (and has been used in 123 the diagnosis of Cycloneuralia as a putative clade) but has cephalic ganglia in the three 124 panarthropod groups (Martin and Mayer 2014; Martín-Durán et al. 2016), as well as the 5 125 nature of the paired versus unpaired nerve cords (Martín-Durán et al. 2016). According to 126 these authors the ancestral nervous system of the Ecdysozoa might have comprised an 127 unpaired ventral nerve cord (seen in Priapulida, Kinorhyncha, Nematoda and 128 Nematomorpha), but the architecture of the brain in the ancestral ecdysozoan remains 129 unclear. The monophyly versus paraphyly of Cycloneuralia (discussed below) is central to 130 the interpretation of whether a collar-shaped or dorsal ganglionar brain is plesiomorphic 131 for Ecdysozoa. 132 From a molecular standpoint, researchers have proposed a series of 133 synapomorphies, such as the identification of ecdysozoan tissue-specific markers, including 134 neural expression of horseradish peroxidase (HRP) immunoreactivity (Haase et al. 2001). 135 Another molecular synapomorphy, a supposed multimeric form of a ß-thymosin gene in 136 arthropods and nematodes to the exclusion of other metazoans (Manuel et al. 2000), has 137 been subsequently refuted (Telford 2004). 138 Ecdysozoans have colonized the land and freshwater independently in multiple 139 lineages (in Nematoda, Nematomorpha, Tardigrada, Onychophora, Chelicerata, Myriapoda, 140 Hexapoda and several other panxcrustacean lineages) between the Cambrian and the 141 Devonian (Rota-Stabelli et al. 2013; Lozano-Fernandez et al. 2016). Clearly, their cuticle has 142 provided them with the physical properties (i.e., avoiding desiccation and providing 143 mechanical support) to conquer the land multiple times—from all other animals, only 144 chordates, platyhelminths, rotifers, annelids, nemerteans and molluscs have been able to 145 terrestrialize. 146 147 Ecdysozoan phylogeny—past and present 148 Despite the vast genomic resources available for many members of Ecdysozoa, relationships 149 within its constituent clade remain in flux (Fig. 1), to the point that many authors use some 6 150 ecdysozoan clades as bona fide, even though no molecular support for them exists, thus 151 relying on morphological hypotheses. Such is the case for Scalidophora (Cephalorhyncha 152 sensu Nielsen) (Fig.1A–C), a putative clade composed of Kinorhyncha, Loricifera and 153 Priapulida (e.g., Edgecombe 2009; Dunn et al. 2014; Martín-Durán et al. 2016). Likewise, 154 whether Scalidophora and Nematoida form the clade Cycloneuralia (= Introverta sensu 155 Nielsen) or a grade, with Nematoida as the sister group of Panarthropoda, is still 156 unresolved. While the challenge to the monophyly of Cycloneuralia is mostly molecular, 157 some morphological inconsistencies are noteworthy. For example, the “typical” 158 cycloneuralian brain, a ring neuropil with anteriorly and posteriorly positioned neuronal 159 somata, is shared by nematodes, kinorhynchs, loriciferans and priapulans, but not 160 nematomorphs (Schmidt-Rhaesa and Rothe 2014). 161 The Cycloneuralia and Panarthropoda controversies hinge on the question of 162 nematodes and tardigrades and their attraction. In the context of Panarthropoda, 163 tardigrades are sometimes believed to be the sister group of arthropods (see Yang et al. 164 2015 for a recent cladistic analysis), mostly due to similarities in the ganglionar peripheral 165 nervous system (Mayer et al. 2013)—as opposed to that of onychophorans. A tardigrade- 166 arthropod clade (Tactopoda) is also recovered in some cladistic analyses coding for a broad 167 range of fossils (Smith and Caron 2015; Yang et al. 2015), although the signal comes 168 principally from characters of the nerve cord in extant taxa. However, a series of 169 phylogenomic analyses have cast some doubt about the membership of Tardigrada in 170 Panarthropoda. Several studies have found a relationship of Tardigrada to Nematoida2 171 (Hejnol et al. 2009; Borner et al. 2014), or recovered either that grouping or Panarthropoda 172 under different analytical conditions (Dunn et al. 2008). Other studies suggest that the 2 This debate is often discussed as a nematode–tardigrade relationship, but this is not entirely precise, as several studies excluded nematomorphs (e.g., Borner et al. 2014). The monophyly of Nematoida is generally well supported, and thus we should refer to a nematoid–tardigrade relationship, although in a few studies tardigrades nested within nematoids. 7 173 tardigrade-nematoid group is due to a long-branch attraction artifact (Campbell et al. 2011; 174 Rota-Stabelli et al. 2013) and instead recovered Panarthropoda as a clade (Pisani et al. 175 2013) when using better models of evolution. The debate is not settled, as many of these 176 studies relied on old ESTs and newer analyses based on genome data or new 177 transcriptomes have found the nematode–tardigrade grouping (Laumer et al. 2015), 178 although the small number of arthropods included in the sample (designed to resolve other 179 parts of the protostome tree) lessens the impact of this result. 180 Molecular analyses excluding Loricifera have supported a sister group relationship 181 of Scalidophora to Nematoida and Panarthropoda (e.g., Petrov and Vladychenskaya 2005; 182 Mallatt and Giribet 2006; Campbell et al. 2011; Pisani et al. 2013; Rota-Stabelli et al. 2013; 183 Borner et al. 2014), or have favored a sister group relationship of Priapulida to the 184 remaining ecdysozoans (Hejnol et al. 2009). Analyses contradicting Scalidophora place 185 Loricifera closer to Nematomorpha than to Kinorhyncha and Priapulida (Sørensen et al. 186 2008), or left Loricifera largely unresolved (Park et al. 2006), although these early analyses 187 were based on just one or two markers. The first phylogenomic analysis to include data on 188 Loricifera places Armorloricus elegans with Priapulida, albeit without significant support 189 (Laumer et al. 2015), and this study lacked data on Kinorhyncha and taxonomic sampling 190 was not designed around Ecdysozoa. 191 The issues with Loricifera noted above largely involve limited molecular sampling to 192 date. In contrast, the debate about the position of tardigrades in ecdysozoan phylogeny 193 involves incongruence between well sampled datasets. Tardigrades have been placed in 194 Panarthropoda using a plethora of morphological characters, as well as in several molecular 195 analyses designed to counter long branch attraction, and based on a novel microRNA 196 (Campbell et al. 2011), but, as discussed above, they are also often drawn to Nematoida in 197 molecular analyses (Yoshida et al. 2017). In contrast to the unstable relationships of 8 198 tardigrades, however, in most cases Onychophora have stabilized as the sister group of 199 Arthropoda (e.g., Hejnol et al. 2009; Campbell et al. 2011; Rota-Stabelli et al. 2013; Borner et 200 al. 2014), a relationship that contradicts the Tactopoda hypothesis. 201 Clearly, further resolution of ecdysozoan relationships is needed, as genomic and 202 transcriptomic resources are still limited for Loricifera, Kinorhyncha, Priapulida and 203 Nematomorpha. Major efforts should be directed towards resolving the Cycloneuralia and 204 Scalidophora questions that presently render the deep splits in Ecdysozoa ambiguous, but 205 also towards more refined analytical treatment of data, including improved models of 206 evolution. We thus favor, for the time-being, the partially unresolved phylogeny presented 207 in Figure 1D until some of these most unstable taxa are available and analyses targeting a 208 well thought-out set of genes provide convincing results. 209 Even defining panarthropods morphologically is less straightforward than it might 210 appear, as most characters typically used in textbooks are absent in one of the three phyla. 211 They all have paired ventrolateral segmental appendages with terminal claws, but the 212 nature of these appendages differs among them. Only arthropods have undergone a true 213 arthropodization process, with both segmental sclerites and appendage segments 214 cuticularized and separated by arthrodial membranes. In spite of this, at least 215 onychophorans and arthropods share the same general patterns of gap gene expression 216 along the proximo-distal axis of the appendages (Janssen and Budd 2010; Janssen et al. 217 2015); these data are not yet known for tardigrades. Likewise, all three groups (tardigrades, 218 onychophorans and arthropods) have a ganglionar supraesophageal brain, but that of 219 tardigrades is composed of a single segment (Gross and Mayer 2015), that of 220 onychophorans of two, protocerebrum and deutocerebrum (Mayer et al. 2010), while 221 arthropods have three, protocerebrum, deutocerebrum and tritocerebrum. The ventral 222 nerve cords of these three groups also differ greatly, with a paired ganglionated nerve cord 9 223 in tardigrades and arthropods versus a lack of segmental ganglia in onychophorans (Martin 224 et al. 2017). This is also concomitant which their external appearance, as onychophorans 225 instead of external segments show an annulated cuticle. Nevertheless, the segment polarity 226 protein engrailed is expressed in the posterior ectoderm of developing segments in each of 227 the three panarthropod groups, suggesting that it plays a common role in establishing 228 segmental boundaries (Gabriel and Goldstein 2007) and can be interpreted as an 229 autapomorphy related to panarthropod segmentation. While segmented mesoderm and a 230 mixocoel have also been proposed as synapomorphies for Panarthropoda (Nielsen 2012), 231 these are not observed in tardigrades. 232 233 Ecdysozoan genomics 234 Ecdysozoan genomics got an early start, as the nematode Caenorhabditis elegans was the 235 first published animal genome (C._elegans_Sequencing_Consortium 1998), to be followed by 236 that of Drosophila melanogaster (Adams et al. 2000). Both appeared before the first drafts of 237 the human genome, attesting to the importance of these two ecdysozoans as model 238 organisms. Since then, more than a hundred ecdysozoan genomes from different species 239 have been published (Dunn and Ryan 2015), and thousands more have been sequenced. No 240 other animal clade except perhaps for vertebrates has such genomic resources. 241 Additionally, high-coverage transcriptomes are now available for virtually every major 242 ecdysozoan lineage (orders or equivalent) (e.g., Misof et al. 2014; Sharma et al. 2014; Wang 243 et al. 2014; Laumer et al. 2015; Fernández et al. 2016; Kocot et al. 2017; Schwentner et al. 244 2017), although many have yet to make it into publication. 245 Tardigrade genomics recently erupted in the scientific debate as an unusual case of 246 massive horizontal gene transfer in the species Hypsibius dujardini (Boothby et al. 2015), to 247 be almost immediately refuted (Koutsovoulos et al. 2016). However, both in H. dujardini 10 248 and Ramazzottius varieornatus a small proportion (ca. 1-2%) of horizontal gene transfer 249 seems justified (Hashimoto et al. 2016; Yoshida et al. 2017). In the latter species, there is 250 also a loss of gene pathways that promote stress damage, expansion of gene families related 251 to ameliorating damage, and evolution and high expression of novel tardigrade-unique 252 proteins (Hashimoto et al. 2016). The proteome of the tardigrade Milnesium tardigradum 253 has been investigated in order to better understand stress pathways (Schokraie et al. 2010; 254 Förster et al. 2012). More recently, differential gene expression between hydrated and 255 dehydrated stages and transition to and from the tun state (the state shown during 256 anhydrobiosis) have shown interesting patterns (e.g., down-regulation of several proteins 257 of the DNA replication and translational machinery and protein degradation) during 258 metabolic shutdown when entering anhydrobiosis (Wang et al. 2014). 259 To date, a single unpublished genome is available for Priapulus caudatus (GenBank 260 accession # NW_014577062), due to recent interest in priapulans as model organisms for 261 understanding early ecdysozoan evolution. Transcriptomic resources are also rather limited 262 for priapulans, with just a few published transcriptomes (Borner et al. 2014; Laumer et al. 263 2015), and EST libraries (Dunn et al. 2008) available. 264 The first sequences for an onychophoran genome (Euperipatoides rowelli) are 265 publicly available (https://www.hgsc.bcm.edu/arthropods/velvet-worm-genome-project), 266 but no genome annotation has yet been produced. Transcriptomic resources have bloomed 267 in recent years, although Illumina-based transcriptomes have only recently been produced 268 (Fernández et al. 2014). Additional transcriptomes are now being generated to investigate 269 the phylogenetic position of onychophorans with respect to arthropods and tardigrades and 270 for developmental research (e.g., Franke et al. 2015). 271 Complete mitochondrial genomes are however available for both onychophoran 272 families (Podsiadlowski et al. 2008; Braband et al. 2010a; Braband et al. 2010b; Segovia et 11 273 al. 2011). These studies indicate that the mitochondrial genome of velvet worms shows 274 major rearrangements and extreme mitochondrial tRNA editing (Segovia et al. 2011), which 275 seems to have persisted through the evolution of the group. 276 Little is known about kinorhynch nuclear genomes, with no size estimate or 277 sequence currently available. Only recently two mitochondrial genomes have been 278 published (Popova et al. 2016)—Echinoderes svetlanae (Cyclorhagida) and Pycnophyes 279 kielensis (Allomalorhagida). Their mitochondrial genomes are circular molecules 280 approximately 15 Kbp in size, with the typical metazoan complement of 37 genes, which are 281 all positioned on the major strand, but the gene order is distinct and unique among 282 Ecdysozoa (Popova et al. 2016), including duplicated methionine tRNA genes. 283 Other than a relatively low quality transcriptome of Armorloricus elegans (Laumer 284 et al. 2015), little is known about the nuclear genome of loriciferans. No information is 285 available for any mitochondrial gene, being probably the only animal phylum without even 286 a single sequence of cytochrome c oxidase subunit I—the so-called “universal barcode” 287 available for all other animals. In addition, a lack of mitochondria has been reported in some 288 species (Danovaro et al. 2010). 289 290 Insights from Cambrian fossils 291 Even discounting the arthropods that are the most common and diverse Cambrian fossils, 292 the Cambrian fossil record of ecdysozoans is spectacular, with several putative basal 293 lineages reaching a peak of diversity at the time (see Maas 2013; for a synopsis of Paleozoic 294 vermiform ecdysozoans). Much of the Cambrian cycloneuralian diversity is represented by 295 Paleoscolecida (Harvey et al. 2010), a group of often large-bodied worms that have a high 296 preservation potential because of their robust, annulated cuticle (Fig. 2A-C). Their cuticular 297 sclerites (Fig. 2C) have an extensive microfossil record when preserved disarticulated from 12 298 their scleritome. Burgess Shale-type compression fossils (Fig. 2A) as well as three- 299 dimensionally preserved, secondarily phosphatized Orsten fossils allow the sclerites to be 300 associated with both the overall cuticular structure as well as other body parts, including 301 paired terminal hooks and an introvert that bears radially arranged spines (Maas 2013). 302 The posterior hooks (Fig. 2B) had been cited as a character indicating affinities to 303 Nematomorpha (Hou and Bergström 1994), and a system of large, helically wound cross- 304 wise fibers in the innermost layer of the cuticle is also comparable to nematoids (Harvey et 305 al. 2010). Numerous phylogenetic analyses have tackled the systematic position of 306 palaeoscolecids and other vermiform ecdysozoans that are not obviously crown-group 307 members of living phyla (see Harvey et al. 2010; Wills et al. 2012; Liu et al. 2014; Zhang et 308 al. 2015 for recent versions). The controversies noted above regarding higher level 309 systematics of Ecdysozoa, notably whether or not Cycloneuralia is a mono- or paraphyletic 310 with respect to Panarthropoda as well as the status of Scalidophora, affect the classification 311 of the fossils. That is, although their introvert morphology may attest to cycloneuralian 312 affinities, this may simply be a plesiomorphic character for Ecdysozoa. In different analyses, 313 palaeoscolecids are variably allied with nematoids or with priapulans, the fossils being 314 sensitive to taxon sampling and character weighting. 315 Numerous other Cambrian vermiform ecdysozoans are known from exceptionally 316 preserved compression fossils. The Burgess Shale species Ottoia prolifica, for example, is 317 known from thousands of specimens that permit details of the eversion of the introvert to 318 be documented (Fig. 2F) (Conway Morris 1977) and gut contents reveal the diversity of 319 prey that it ingested (Vannier 2012). Ottoia is usually resolved in phylogenetic analyses as a 320 stem-group priapulan, but like many of the fossils its position has been labile within 321 “Cycloneuralia”. The Cambrian vermiform ecdysozoans include some distinctive ecologies, 322 including tube-swelling forms such as Selkirkia (Conway Morris 1977). 13 323 With regards to the timing of ecdysozoan diversifcation, the fossil record indicates 324 that some extant phyla were likely represented by their crown groups in the Cambrian. This 325 is the case, for example, for Loricifera, of which Eolorica deadwoodensis, is a late Furongian 326 (late Cambrian) member (Harvey and Butterfield 2017). This species, with typical 327 meiobenthic size and morphology (Fig. 2E), exhibits the characteristic high number of 328 scalids of Loricifera, typical spinoscalid form, and a loricate body. The latter particularly 329 resembles the members of the extant family Pliciloricidae in its large number of plicae, and 330 is consistent with Eolorica being a crown group loriciferan. Likewise, phylogenetic analyses 331 have placed some early Cambrian priapulans in crown-group Priapulida (Ma et al. 2014), 332 and Arthropoda is likewise represented in the early Cambrian (ca 519 Ma) by crown-group 333 taxa (Edgecombe, this volume). 334 Fossil taxa provide combinations of arthropod and cycloneuralian characters not 335 observed in any living ecdysozoan. For example, a radial mouth composed of overlapping 336 plates and radially aranged, scalid-like pharyngeal teeth in such giant stem-group 337 arthropods as the early Cambrian Pambdelurion are interpreted as plesiomorphies shared 338 by Panarthropoda and “cycloneuralians”, and thus characters of the Ecdysozoa as a whole 339 (Edgecombe 2009; Vinther et al. 2016). Likewise, the Cambrian lobopodian Hallucigenia, 340 which has been interpreted as a stem-group onychophoran (Smith and Ortega-Hernández 341 2014) or a stem-group panarthropod (Caron and Aria 2017), has radially arranged 342 circumoral lamellae and pharyngeal teeth that compare with putative homologues in 343 tardigrades and cycloneuralians and accordingly cited as possible autapomorphies of 344 Ecdysozoa (Smith and Caron 2015). Cambrian lobopodians are resolved in phylogenetic 345 analyses as an aggregation of stem-group tardigrades, onychophorans and arthropods 346 (Yang et al. 2015; Caron and Aria 2017). Based on the resulting trees, the most recent 347 common ancestor of extant Panarthropoda was a macroscopic lobopodian with 14 348 heteronomous body annulation, an anteriorly-facing mouth with radial circumoral papillae, 349 and paired dorsolateral epidermal structures in segmental association with lobopodous 350 limbs (Smith and Ortega-Hernández 2014). 351 352 The future of ecdysozoan phylogenetics 353 The incredible genomic resources available for ecdysozoans hold a promise for a well354 resolved phylogeny, although a major issue seems to be a highly heterogeneous rate of 355 evolution across lineages as well as large variation in genome size and content, as for 356 example, some nematodes have among the smallest genomes (Burke et al. 2015). Previous 357 limitations of size for genomic work, especially in loriciferans, will soon no longer be an 358 issue with developing single cell genomic techniques (Zheng et al. 2017). Yet, placing 359 certain taxa continues to be nearly intractable with existing phylogenetic methods (Simion 360 et al. 2017), and nematodes, tardigrades and most probably also loriciferans, do not seem to 361 be immune to some of these biases. The bright side is that we have yet to test their 362 relationships with improved taxon sampling and modern molecular 363 (genomic/transcriptomic) data, and the constant discovery of new fossils (e.g., Harvey and 364 Butterfield 2017) will continue to contribute towards a better understanding of the stems 365 leading to the major ecdysozoan clades (see Edgecombe, this volume). 366 367 Acknowledgements 368 This contribution is derived from a SICB symposium “ The Evolution of Arthropod Body 369 Plans – Integrating Phylogeny, Fossils and Development” organized by Ariel Chipman and 370 Doug Erwin. Three referees are acknowledged for their valuable comments. 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Massively parallel digital transcriptional profiling of single cells. Nat Commun 8:14049. Zrzavý J, Mihulka S, Kepka P, Bezdek A, Tietz D. 1998. Phylogeny of the Metazoa based on morphological and 18S ribosomal DNA evidence. Cladistics 14:249-85. 21 695 Fig. 1. Summary of selected ecdysozoan phylogenies from (A) Nielsen (2012); and other 696 less resolved versions presented in recent textbooks and reviews: (B) Dunn et al. (2014); 697 Brusca and Giribet (2016); Giribet (2016b); (C) Piper (2013); (D) Giribet (2016a); version 698 D, highlighted in grey is the version that we currently support based on all available data. 699 Selection of of metazoan phylogenies based on analysis of: (E) EST data (Campbell et al. 700 2011); (F) transcriptomes (Borner et al. 2014); (G) ESTs (Dunn et al. 2008); (H) ESTs 701 (Hejnol et al. 2009); (I) transcriptomes (Laumer et al. 2015). 702 703 Fig. 2. Exceptionally preserved Cambrian and Ordovician fossil Ecdysozoa. A, B, Wronascolex 704 antiquus, a palaeoscolecid worm from the early Cambrian Emu Bay Shale, Australia; A, 705 mostly complete specimen, scale 1 cm; B, paired terminal hooks, scale 2 mm; C, Gamascolex 706 vanroyi, a palaeoscolecid from the Late Ordovician of Morocco. Scanning electron 707 micrograph showing rows of plates on the cuticular annulations, scale 0. 5mm. Image 708 courtesy of Diego García-Bellido; D, Aysheaia pedunculata, a lobopodian from the middle 709 Cambrian Burgess Shale, Canada, scale 2.5 mm; E, Eolorica deadwoodensis, a loriciferan 710 preserved as a Small Carbonaceous Fossil from the late Cambrian of Canada, scale 0.25 mm. 711 Image courtesy of Tom Harvey and Nick Butterfield; F, Ottoia prolifica, a cycloneuralian 712 from the Burgess Shale, Canada, scale 5 mm. Images D, F courtesy of Xiaoya Ma. 22 A Priapulida Kinorhyncha Loricifera Nematoda Scalidophora Nematomorpha Tardigrada Nematoida Cycloneuralia Onychophora Arthropoda Panarthropoda B CD E Summary trees FG H I Published phylogenetic trees