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Opsin evolution in the Ambulacraria. D'Aniello, S; Delroisse, J; Valero-Gracia, A; Lowe, EK; Byrne, M; Cannon, JT; Halanych, KM; Elphick, MR; Mallefet, J; Kaul-Strehlow, S; Lowe, CJ; Flammang, P; Ullrich-Lüter, E; Wanninger, A; Arnone, MI

© 2015 The Authors. CC-BY-NC-ND

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Opsin evolution in the Ambulacraria 1 2 3 D Aniello S1,§,*, Delroisse J2,3,§, Valero-Gracia A1, Lowe EK1,4, Byrne M5, Cannon 4 ’ 5 6 6,7 6 3 8 9 10 7 JT , Halanych KM , Elphick MR , Mallefet J , Kaul-Strehlow S , Lowe CJ , 8 9 Flammang P2, Ullrich-Lüter E11, Wanninger A12 and Arnone MI1 10 11 12

13 1 14 Department of Biology and Evolution of Marine Organisms, Stazione Zoologica Anton Dohrn, Villa Comunale, 15 80121 Napoli, Italy. 16 2 Biology of Marine Organisms and Biomimetics, Research Institute for Biosciences, University of Mons, Avenue du 17 18 Champs de Mars 6, 7000 Mons, Belgium. 19 3 School of Biological & Chemical Sciences, Queen Mary University of London, London, E1 4NS, UK. 20 4 BEACON Center for the Study of Evolution in Action, Michigan State University, East Lansing, Michigan, USA 21 22 5 Schools of Medical and Biological Sciences, The University of Sydney, Sydney, NSW, Australia. 23 6 Department of Biological Sciences and Molette Biology Laboratory for Environmental and Climate Change 24 25 Studies, Auburn University, Auburn, USA. 26 7 Department of , Naturhistoriska Riksmuseet, Stockholm, Sweden; Friday Harbor Laboratories, University 27 28 of Washington, Friday Harbor, WA 98250, USA. 29 8 Laboratory of Marine Biology, Earth and Life Institute, Université Catholique de Louvain, Louvain-La-Neuve, 30 31 Place Croix du Sud 3, bt L7.06.04, 1348 Louvain-la-Neuve, Belgium. 32 9 Department of Molecular Evolution and Development, University of Vienna, Althanstrasse 14, 1090 Vienna, 33 34 Austria. 35 10 Hopkins Marine Station of Stanford University, Pacific Grove, CA 93950, USA. 36 11 Museum fuer Naturkunde Berlin, Invalidenstr 43, 10115 Berlin, Germany. 37 12 38 Department of Integrative Zoology, University of Vienna, Althanstrasse 14, 1090 Vienna, Austria. 39 40 41 42 43 44 45 46 47 48 49 50 § 51 These authors contributed equally to this work. 52 53 54 *Corresponding author: [email protected] 55 56 57 58 59 60 61 62 63 64 1 65 Abstract 1 2 3 Opsins - G-protein coupled receptors involved in photoreception - have been 4 5 extensively studied in the . The present work provides new insights into 6 opsin-based photoreception and photoreceptor cell evolution with a first analysis of 7 8 opsin sequence data for a major clade, the Ambulacraria. Systematic data 9 10 analysis, including for the first time opsin sequences and an expanded 11 12 dataset, led to a robust opsin phylogeny for this cornerstone superphylum. 13 14 Multiple genomic and transcriptomic resources were surveyed to cover each class of 15 16 Hemichordata and Echinodermata. In total, 119 ambulacrarian opsin sequences were 17 found, 22 new sequences in and 97 in (including 67 new 18 19 sequences). We framed the ambulacrarian opsin repertoire within eumetazoan diversity 20 21 by including selected reference opsins from non-ambulacrarians. Our findings 22 23 corroborate the presence of all major ancestral bilaterian opsin groups in Ambulacraria. 24 25 Furthermore, we identified two opsin groups specific to echinoderms. In conclusion, a 26 27 molecular phylogenetic framework for investigating light-perception and 28 photobiological behaviours in marine has been obtained. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 Keywords: Opsin; Photoreceptor Cell Evolution; Ambulacraria; Echinoderm; 54 55 Hemichordate; Phylogeny; Echinopsin. 56 57 Manuscript type: Original data paper. 58 59 60 61 62 63 64 2 65 Introduction 1 2 3 In , the prototypical molecules involved in photoreception and vision are opsin 4 5 proteins (Nilsson, 2004). Opsins are G-protein coupled receptors (GPCR) that consist of 6 an apoprotein plus a covalently bound to a chromophore (11-retinal) (Terakita, 2005). 7 8 The nitrogen atom of the amino group of residue K296, situated in helix VII, binds to 9 10 the retinal molecule through a Schiff-base linkage, forming a double bond with the 11 12 carbon atom at the end of this molecule (Hargrave et al., 1983). Residue K296 is, 13 14 therefore, crucial for light absorption, and its presence or absence can be used as a 15 16 molecular fingerprint to judge whether or not a GPCR is a bona fide opsin. 17 Recent investigations on opsin phylogeny resolved six distinct groups present in 18 19 metazoans: ciliary opsins, rhabdomeric opsins, Go-opsins, neuropsins, peropsins, and 20 21 RGR (RPE-retinal G protein-coupled receptor) opsins (Porter et al., 2012; Feuda et al. 22 23 2012; Terakita et al., 2012). A vast number of opsins are also expressed in non-ocular 24 25 tissues (Porter et al., 2012; Plachetzki et al., 2005; Koyanagi et al., 2005; Terakita et 26 27 al., 2012). 28 With regard to opsin evolution in the deuterostomes, genomic and transcriptomic data 29 30 of a number of have been used to identify and characterize their opsins (e.g. 31 32 Holland et al., 2008; Kusakabe et al., 2001). However, little attention has been paid to 33 34 Ambulacraria, the sister group to all extant chordates, (i.e. , 35 36 urochordates, and , Edgecombe et al., 2011), a key clade to reconstruct the 37 38 opsin set of the common ancestor of extant deuterostomes. 39 The present study integrates opsin sequences from two ambulacrarian sub-lineages: 40 41 enteropneust Hemichordata, (Harrimaniidae, Spengelidae, Ptychoderidae and 42 43 Torquaratoridae), and the pentameral Echinodermata comprising five classes 44 45 (Crinoidea, Ophiuroidea, Asteroidea, Holoturoidea and Echinoidea). 46 47 The phylogenetic relationship of echinoderms and hemichordates as sister groups within 48 Ambulacraria, as shown in Figure 1, was already suggested by Metschnikoff (1881), 49 50 and supported by Nielsen (2012). The monophyly of Ambulacraria is also well 51 52 supported by molecular phylogenetic analyses (Cannon et al., 2014; Telford et al., 53 54 2014). Moreover, Cannon and colleagues showed that the six hemichordate subgroups 55 56 cluster into two monophyletic taxa, Enteropneusta and (Rhabdopleuridae 57 58 and Cephalodiscidae). Finally, Figure 1 conforms to the hypothesis 59 separating the (Echinoidea + Holothuroidea) and the Asterozoa (Asteroidea 60 61 62 63 64 3 65 + Ophiuroidea), which is now well supported by recent molecular phylogenies (Cannon 1 2 et al., 2014; Telford et al., 2014; O’Hara et al., 2014). 3 4 Other than a few structural investigations of eye-like structures in some asteroid species 5 6 (e.g. the optic cushion) and in enteropneust larvae (Brandenburger et al., 1973; 7 8 Nezlin and Yushin, 2004; Braun et al., 2015), the molecular mechanisms of echinoderm 9 10 and hemichordate photoreception remained enigmatic until recently. 11 12 Immunohistochemical studies indicated the presence of a putative rhodopsin in the 13 14 asteroid Asterias forbesi and in the ophiuroid Ophioderma brevispinum (Johnsen, 15 16 1997). Subsequently, Raible et al. (2006) analyzed the ‘rhodopsin-type’ G-protein- 17 18 coupled receptors family in an echinoid genome (Strongylocentrotus purpuratus). They 19 20 predicted six bona fide opsin sequences, four of which were reported independently by 21 22 Burke et al. (2006). Later, Ooka et al. (2010) cloned an “encephalopsin” orthologue in 23 24 25 the Hemicentrotus pulcherrimus. Recently, more opsin sequences have been 26 found in sea urchins (S. purpuratus; Paracentrotus lividus), starfish (Asterias rubens), 27 28 and brittle stars (Ophiocomina nigra, Amphiura filiformis) (Delroisse et al., 2013, 2014, 29 30 2015 a,b ; Ullrich-Lüter et al., 2011, 2013). These studies highlighted the expression of 31 32 ciliary and rhabdomeric opsins in various echinoderm tissues. Also, a large opsin gene 33 34 repertoire was identified in the A. filiformis, pinpointing notable differences 35 with findings from the previously published sea urchin genome (Delroisse ., 2014). 36 et al 37 However, a comprehensive description of opsin diversity in echinoderms is still lacking 38 39 and almost nothing is known about hemichordate opsins. 40 41 Therefore, to characterize and describe the diversity of the opsin family in the 42 43 Ambulacraria, we conducted a detailed analysis of 6 genomic and 24 transcriptomic 44 45 sequence databases. This work represents the first attempt to describe and characterize 46 47 the evolution of the opsin “toolkit” in the ambulacrarian lineage. We performed a 48 49 phylogenetic study using the largest dataset of ambulacrarian opsin sequences to date, 50 51 including representatives of a previously neglected group, Hemichordata. 52 53 54 55 56 Materials and methods 57 58 59 60 Data mining 61 62 63 64 4 65 Strongylocentrotus purpuratus opsins belonging to all the paralogous classes (Supp. 1 2 File 1) were used as starting query sequences for tBLASTx against transcriptomic and 3 genomic databases including public databases (NCBI, JGI, Ensemble, Echinobase 4 5 (www.echinobase.org/), BioInformatique CNRS-UPMC (http://octopus.obs-vlfr.fr/) and 6 7 Genoscope (http://www.genoscope.cns.fr/spip/Generation-de-ressources.html). The 8 9 parameters used across all our tBLASTx searches were the following: Matrix: 10 11 Blosum62; gap penalties: existence: 11; extension: 1; neighboring words threshold: 13; 12 13 window for multiple hits: 40. Additionally, our dataset was further enriched using 14 various unpublished genomic and transcriptomic databases obtained from several 15 16 independent research projects (Suppl. File 1 and Suppl. File 2). This includes 17 18 transcriptomes from adult specimens’ tissues, such as cuverian tubules and integument 19 20 from Holothuria forskali, muscle of Parastichopus californicus, radial nerve from 21 22 Asterias rubens, arms from Labidiaster annulatus, Ophiopsila aranea, Astrotomma 23 24 agassizii and Antedon mediterranea, proboscis from Saccoglossus mereschkowskii and 25 Torquaratorid sp, whole adult body of Leptosynapta clarki and anterior part of the body 26 27 from Harrimaniidae sp and Schizocardium braziliense. Several other transcriptomes 28 29 obtained from embryos or larvae from Paracentrotus lividus, Heliocidaris 30 31 erythrogramma, Eucidaris tribuloides, Parasticopus parvimensis, Saccoglossus 32 33 kowalevskii and Ptychodera flava (Suppl. File 1 and Suppl. File 2) were also screened. 34 35 The raw predicted opsin sequences used in this study are listed in the Suppl. File 3 in 36 fasta format. 37 38 39 40 Alignment and phylogenetic analyses 41 42 Predicted protein alignments were performed with SeaView v4.2.12 (Galtier et al., 43 44 1996; Gouy et al., 2010) using the MUSCLE algorithm (Edgar, 2004). To improve 45 phylogenetic reconstruction, N-terminal and C-terminal ends were trimmed and the 46 47 alignment was manually corrected in order to minimize gaps and eliminate ambiguous 48 49 and misaligned regions. Sequences that were shorter than 60 amino acids were removed 50 51 to avoid bias. However, these could potentially correspond to true opsins and merit 52 53 further study. 54 55 Maximum likelihood analyses (ML) of our dataset were conducted on Michigan State 56 University’s High Performance Computing Cluster using PhyML v3.0 (Guindon and 57 58 Gascuel, 2003), and nodal support assessed with 1000 bootstrap replicates is indicated. 59 60 The alignment is shown in Suppl. File 4 (phylip format) and Suppl. File 5 (image). A 61 62 63 64 5 65 best-fit model analysis was performed using MEGA6 (following the AIC criteria) 1 2 (Tamura et al., 2007; Kumar et al., 2008) and WAG+G+F amino acid substitution 3 model was found to be the best suited (Whelan and Goldman, 2001). Three melatonin 4 5 receptor sequences from S. purpuratus (Echinodermata) and three from Saccoglossus 6 7 kowalevskii (Hemichordata) were chosen as the best outgroup for the opsin phylogeny, 8 9 as previously proposed by Plachetzki et al. (2010) and Feuda et al. (2014). 10 11 12 13 Consensus fingerprint of ambulacrarian opsin groups 14 Ambulacraria opsins were clustered according their estimated position within opsin 15 16 subfamilies and a multiple alignment of a 35 amino-acid long peptide region, including 17 18 the 7th transmembrane with the opsin-specific lysine (K296), was performed 19 20 with SeaView v4.2.12 for each opsin group supported in our . The 21 22 selected region spanned residues 286 to 320 of the Rattus norvegicus rhodopsin 23 24 sequence used as a reference (Palczewski et al., 2000). The consensus sequence was 25 generated on the basis of the alignment for each class of ambulacrarian opsin using 26 27 Geneious®8.1.5. 28 29 30 31 32 33 Results 34 35 36 37 Phylogeny and opsin distribution within ambulacrarian groups 38 Using a collection of both genomic and transcriptomic data (see Materials and Methods 39 40 and Suppl. File 2 for details), a final set of 119 protein sequences, representing 31 41 42 ambulacrarian species, was generated for our phylogenetic reconstruction, which 43 44 included 6 outgroup sequences and 6 human reference opsin sequences (Suppl. File 1 45 46 and Suppl. File 3 for raw predicted protein sequences). The trimmed opsin alignment is 47 48 shown in the Suppl. File 5 (see Suppl. File 4 for the alignment phylip file). We 49 employed maximum likelihood using the WAG+G+F model with melatonin receptors 50 51 as an outgroup. Canonical opsin groups are well supported in our analysis (Figure 2), 52 53 demonstrating the presence of a complex opsin toolkit in Ambulacraria. 54 55 Interestingly, according to our data, two novel groups of opsins were found, which we 56 57 have named echinopsin-A and echinopsin-B groups. Ad hoc BLAST searches against 58 59 several metazoan genomes clearly indicated the absence of these two opsin types 60 61 62 63 64 6 65 outside the echinoderm lineage. The previously identified Sp-opsin2 and Sp-opsin5 1 2 belong to echinopsins-A and echinopsins-B, respectively (Raible et al., 2006). 3 A complete opsin profile including at least one representative of each prototypical opsin 4 5 group (opsin 1-8) was detected in the sea urchin S. purpuratus, but not in Lytechinus 6 7 variegatus or P. lividus. The genomes of the latter two species have not yet been 8 9 comprehensively sequenced and annotated, and therefore some opsin genes may be 10 11 missing due to incomplete sequence coverage. With the exception of echinopsin-B, a 12 13 complete opsin profile was found in the genome sequence data of the starfish Patiria 14 miniata. The starfish A. rubens radial nerve transcriptome also contained several opsins, 15 16 including ciliary, Go-, RGR-opsins. 17 18 Surprisingly, rhabdomeric and Go-opsins do not seem to be present in hemichordates in 19 20 our dataset. However, this requires confirmation through more extensive taxonomic 21 22 sampling of hemichordate sequence data because, at present, only one hemichordate 23 24 genome has been fully sequenced (S. kowalevskii). In several opsin groups we observed 25 lineage-specific duplications: two opsins in P. miniata and A. rubens; five neuropsins in 26 27 S. kowalevskii; four r-opsins in L. annulatus and six r-opsins in A. filiformis; two Go- 28 29 opsins in the echinoids L. variegatus, S. purpuratus and Heliocidaris erythrogramma. 30 31 Nevertheless, some of these molecules present a short overlapping sequence therefore 32 33 we cannot exclude that they could be part of unique genes and therefore to overestimate 34 35 their number. 36 37 38 Alignment of the transmembrane domain and opsin fingerprint 39 40 In order to build a consensus fingerprint to distinguish the various ambulacrarian opsin 41 th 42 groups, the 7 transmembrane domain and C-terminal tail region of our sequence 43 44 dataset were aligned and a graphical representation was generated (Fig. 3). All 45 sequences were characterized by the general structure of G protein-coupled receptors 46 47 (GPCRs) comprising seven transmembrane (TM) domains. Numerous residues 48 49 characteristic of opsins are present in the opsin sequences of A. filiformis. However, as 50 51 several sequences are partial, not all characteristic residues could be detected in all 52 53 sequences. Most of the opsin sequences also contained the highly conserved lysine 54 55 residue (equivalent to K296 of the R. norvegicus rhodopsin) critical for Schiff base 56 linkage formed with retinal, except three sea-urchin peropsins (Sp-opsin 6, Pl-opsin 6, 57 58 Lv-opsin 6) in which it is substituted by a glutamate (E). The dipeptide NP (position 59 60 302-303 of the R. norvegicus rhodopsin sequence) is also highly conserved among all 61 62 63 64 7 65 the subfamilies except in peropsins (N/HP) and RGR-opsins, which show divergence in 1 2 these residues (also rhabdomeric opsins to a lesser extent). Amino-acid conservation for 3 each opsin group from our phylogenetic analysis is shown in Figure 3. Ambulacrarian 4 5 c-opsins, r-opsins and echinopsins-A displayed a highly conserved tyrosine (Y306). 6 7 Conversely, the histidine (H310) appears distinctive of the ambulacrarian r-opsins 8 9 (Figure 3) and r-opsins in general (human melanopsin, octopus rhodopsin and 10 11 Drosophila Rh1-opsin). In our dataset the tripeptide SSS, positioned at residues 309- 12 13 402 of the reference protein, is a distinctive feature of ambulacrarian Go-opsins. 14 These representations will be particularly useful in future studies in support of 15 16 phylogenetic analysis to assign novel, unknown sequences to lineage-specific opsin 17 18 groups. 19 20 21 22 23 24 Discussion 25 26 27 Our phylogenetic analyses showed ambulacrarian opsin sequences to be represented in 28 29 all six prototypical bilaterian opsin groups: ciliary opsins, rhabdomeric opsins, 30 31 neuropsins, Go-opsins, peropsins and RGR-opsins (Fig. 4). In addition we confirmed 32 33 the presence of two novel echinoderm-specific opsin groups, which we have named 34 35 echinopsins (echinopsin-A and echinopsin-B). These novel groups of opsins, which 36 37 were found only in Echinoidea, Ophiuroidea and Asteroidea, respectively cluster as a 38 sister group of all other opsins and as a sister group of all opsins except Echinopsins-A 39 40 and ciliary opsins (Fig.4). A deeper analysis of these groups of proteins, including more 41 42 hemichordate opsin sequences, is needed in order to determine if they represent an 43 44 echinoderm or ambulacrarian novelty. 45 46 Our analysis failed to reveal a rhabdomeric opsin (r-opsin) in hemichordates. The 47 48 absence of such an opsin type is surprising because many enteropneust tornaria larvae 49 possess eyespots that bear photoreceptors with clear microvillar surface enlargement 50 51 (Brandenburger et al., 1973; Nezlin and Yushin, 2004; Braun et al., 2015). So far, 52 53 photoreception in microvillar photoreceptor cell types has been demonstrated to 54 55 generally deploy opsins of the so-called rhabdomeric type (r-opsins), although co- 56 57 expression of other opsin types in microvillar/rhabdomeric photoreceptors has been 58 59 shown in recent studies (Randel et al., 2013). However, although our analysis reveals no 60 such opsin in any of the examined enteropneust species, it should be noted that genomic 61 62 63 64 8 65 information is only available from the direct developer S. kowalevskii, which does not 1 2 have a larval (tornarian) stage in its life cycle. Moreover, most of hemichordate 3 transcriptomes in our study were generated using adult tissues; it is therefore possible 4 5 that the absence of r-opsin in this group of animals is due to a limitation of data 6 7 availability from this understudied group of animals. 8 9 In contrast to the lack of r-opsins in enteropneusts, our analyses showed several cases of 10 11 opsin gene duplication. Obviously in some instances the locus of duplication prompted 12 13 a large expansion of the gene family, as is the case of the five neuropsins found in S. 14 kowalevskii, and the six rhabdomeric opsins in A. filiformis, with the latter previously 15 16 described by Delroisse et al. (2014). However, the fragmentary information about these 17 18 duplicates makes it difficult to predict the exact number of functional opsin proteins in 19 20 Ambulacraria. Whether or not these duplicated genes have sub-functionalized roles 21 22 should be experimentally investigated by knock-out or silencing experiments. 23 24 Until recently, under-representation of many taxonomic groups in comparative studies 25 of photoreceptor evolution has hidden the real extent of opsin diversity (Porter et al., 26 27 2012; Feuda et al., 2014). As more opsins have been characterized, these sequences 28 29 have been classified into narrow pre-defined groups (e.g. Group 4 opsins), implying 30 31 theoretical functional similarities that might not always be correct (Shichida and 32 33 Matsuyama, 2009). At present, however, the rapidly increasing availability of entire 34 35 genomes and transcriptomes provides a large number of sequences for investigating the 36 evolution and functional diversity of the opsin family in greater detail. Likewise, our 37 38 detailed phylogenetic analyses of ambulacrarian opsins not only provide a better 39 40 understanding of opsin evolution in general, but are also essential for future 41 42 photoreceptor studies elucidating the evolution of opsin functions. 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 9 65

1 Figures 2 3 4 5 Figure 1. Ambulacrarian phylogenetic relationships and their adult forms. 6 7 The Ambulacraria consist of two groups: Hemichordata, bilateral animals subdivided in 8 six clades: Cephalodiscidae, Rhabdopleuridae, Harrimaniidae, Spengelidae, 9 10 Ptychoderidae and Torquaratoridae, and the pentameral Echinodermata, comprising: 11 12 Crinoidea, Ophiuroidea, Asteroidea, Holoturoidea and Echinoidea. For each class there 13 14 is a representation of the adult body plan. The numbers represented on the figure 15 16 correspond to the two hemichordate subgroups: 1. Pterobranchia and 2. Enteropneusta, 17 18 and the two echinoderm subgroups 3. Eleutherozoa and 4. . 19 20 21 Figure 2. Phylogenetic reconstruction of ambulacrarian opsins. 22 23 119 opsin from 31 different ambulacrarian species cluster in eight highly supported 24 25 groups in this maximum likelihood (ML) based analysis. R-opsins in blue, c-opsins in 26 27 red, Go-opsins in green, neuropsins in purple, peropsins in yellow and RGR-opsin in 28 29 orange. Visualization was generated with Figtree. 30 31 32 Figure 3. Consensus sequences of different opsin groups. 33 34 Graphical representations of opsin amino acid patterns within the multiple alignments of 35 th th 36 the 7 transmembrane domain and the protein G linkage site. The 7 transmembrane 37 38 domain is highlighted in green in the tridimensional representation of a typical opsin 39 receptor. Alignment is limited to the highly conserved regions including the opsin- 40 41 specific lysine residue and the “NPxxY(x)6F” pattern. The lysine residue involved in 42 43 the Schiff base formation - equivalent to K296 of the R. norvegicus rhodopsin - is 44 45 present in position 10. The pattern “NPxxY(x)6F” (position 302-313 of the R. 46 47 norvegicus rhodopsin sequence) is present in position 17-28. The size of each amino 48 49 acid indicates the probability to find this specific amino acid for the considered position. 50 Amino acid patterns of Melatonin receptors used as an outgroup in the phylogenetic 51 52 analysis is also presented. 53 54 55 56 Figure 4. Opsin distribution within the investigated ambulacraria species. 57 58 For each species the number of opsin belonging to classical groups were reported. 59 60 Those species for which no opsins were find are not reported in the table (for additional 61 62 63 64 10 65 informations see Suppl. File 1). Species for which the genome data is available are in 1 2 bold. 3 4 5 Supplementary Figure 1. List of all the ambulacraria species used in our analysis 6 7 and the opsin gene content per species. 8 9 10 11 Supplementary Figure 2. List of the ambulacraria transcriptomes surveyed in our 12 13 analysis. The available information regarding the transcriptomes used is provided and 14 the RNA source material is detailed. 15 16 17 18 Supplementary Figure 3. Opsin fasta file. Predicted opsin proteins used in the 19 20 phylogenetic analysis are listed in fasta format. 21 22 23 24 Supplementary Figure 4. The phylip version of the trimmed alignment used in the 25 study. 26 27 28 29 Supplementary Figure 5. Trimmed alignment supporting the phylogenetic tree 30 31 shown in Figure 2. 32 33 Trimmed alignment of all deduced amino acid sequences of Ambulacraria opsins. The 34 35 alignment mainly contain the “TM cores” of the opsins. Non-conserved N-terminus and 36 C-terminus ends were trimmed. The Schiff base equivalent to the lysine residue in the 37 38 position 296 of the R. norvegicus rhodopsin – is indicated by an asteriscs in the 39 40 alignment. Alignment performed in Seaview and edited in Geneious®8.1.5. 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 11 65

1 Aknowledgements 2 3 The authors would like to thank Santiago Valero-Medranda for the illustrations in 4 5 Figure 1; Prof. Andy Cameron, Dr. Joel Smith, the staff of Echinobase for giving access 6 7 to various echinoderm sequences, and Thierry Lepage and Genoscope P. lividus Project 8 9 N°17. Paola Oliveri, UCL (UK) and Yi-Hsien Su, Academia Sinica of Taipei (Taiwan) 10 11 for their suggestions in the early phase of the project. The authors would like to thank 12 13 Elise Hennebert, Mélanie Demeuldre and Akihito Omori for providing access to the A. 14 15 rubens tube foot transcriptome, the H. forskali cuverian tubule transcriptome and the 16 17 Oxycomanthus japonicus embryo transcriptome respectively. S. D’Aniello is supported 18 by a Marie Curie Career Integration Grant, FP7-PEOPLE-2011-CIG (grant no. PCIG09- 19 20 GA-2011-293871). J. Delroisse is currently supported by BBSRC grant no. 21 22 BB/M001644/1 awarded to M.R. Elphick (QMUL). A. Valero-Gracia is supported by 23 24 the Marie Curie ITN ‘Neptune’ (grant no. 317172, PI: M.I. Arnone). This work is 25 26 supported in part by the “National funds for research” (FNRS) of Belgium (FRFC grant 27 28 no. 2.4590.11). K.M. Halanych and J.T Cannon hemichordate's transcriptomes were 29 funded by the USA National Science Foundation (grant no. DEB-0816892). E. Ullrich- 30 31 Lüter is supported by a grant of the German Research foundation (DFG) grant no: UL 32 33 428/2-1. M. Byrne is supported by that Australian Research Council (DP120102849). S. 34 35 Kaul-Strehlow is supported by a grant of the Austrian Science Fund (FWF, M-1485). 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 12 65 References 1 2 3 4 Arendt, D., 2008. The evolution of cell types in animals: emerging principles from 5 6 molecular studies. Nat. Rev. Genet. 9, 868-82. 7 8 9 Angueyra, J.M., Pulido, C., Malagón, G., Nasi, E., Gomez, M.P., 2012. 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Evol. 28 18, 691-699. 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 18 65 Figure 1 AMBULACRARIA ECHINODERMATA HEMICHORDATA 3 2 4 1 Cephalodiscidae Rhabdopleuridae Ptychoderidae Spengelidae Harrimaniidae Crinoidea Holothuroidea Echinoidea Ophiuroidea Asteroidea Torquaratoridae Figure 2 505 A.filiformis ops2 978 S.purpuratus ops2 E.tribuloides ops2 Echinopsins A 653 H.erythrogramma ops2 293 Homo Encephalopsin Homo Rhodopsin 982 P.flava ops1 B. aurantiacus ops1 401 775 A.rubens ops1.2 P.miniata ops1.2.A 563 332 A.filiformis ops1 795 P.miniata ops1.2.B 826 P.lividus ops1.2 792 L.variegatus ops1 141 S.purpuratus ops1 Ciliary opsins H.pulcherrimus ops1 468 P.lividus ops1.1 511 E.tribuloides ops1 L.clarki ops1 272 H.forskali ops1 579 H.glaberrima ops1 878 P.parvimensis ops1 P.californicus ops1 A.rubens ops1.1 593 P.miniata ops1.1 210 S.purpuratus ops5 1000 S.droebachiensis ops5 S.intermedius ops5 901 P.lividus ops5 L.variegatus ops5 Echinopsins B 798 A.filiformis ops5 P.miniata ops5 L.annulatus ops4.2 1000 L.annulatus ops4.3 542 L.annulatus ops4.4 L.annulatus ops4.1 Homo_Melanopsin 559 H.forskali ops4 999 H.glaberrima ops4 P.parvimensis ops4 436 L.variegatus ops4 22 E.tribuloides ops4 914 980 S.intermedius ops4 S.purpuratus ops4 P.lividus ops4 Rhabdomeric opsins 472 942 A.filiformis ops4.3 224 A.filiformis ops4.1 958 O.aranea ops4 280 A.filiformis ops4.2 A.agassizii ops4 953 339 A.filiformis ops4.6 998 825 A.filiformis ops4.5 A.filiformis ops4.4 701 A.rubens ops4 P.miniata ops4 948 A.mediterranea ops4 F.serratissima ops4 23 Homo Neuropsin 262 B. aurantiacus ops8 Schizocardium sp ops8 748 212 687 S.kowalevskii ops8.4 739 S.kowalevskii ops8.1 990 467 S.kowalevskii ops8.2 388 S.kowalevskii ops 8.5 S.kowalevskii ops8.3 521 P.parvimensis ops8 Neuropsins P.flava ops8 407 670 A.filiformis ops8.1 283 P.miniata ops8 A.filiformis ops8.2 696 H.erythrogramma ops8 540 987 S.purpuratus ops8 E.tribuloides ops8 123 A.filiformis ops3 998 A.rubens ops3.1.A 1000 990 A.rubens ops3.1.B P.miniata ops3.1 P.parvimensis ops3.1 387 H. erythrogramma ops3.1.B 384 L.variegatus ops3.1.A H. erythrogramma ops3.1.A 390 P.lividus ops3.1.A Go-opsins 784 S.purpuratus ops3.1 170 P.lividus ops3.1.B L.variegatus ops3.1.B E.tribuloides ops3 985 P.lividus ops3.2 423 L.variegatus ops3.2 770 S.purpuratus ops3.2 Homo Peropsin 999 S.kowalevskii ops6 532 P.flava ops6 999 P.lividus ops6 S.purpuratus ops6 121 721 577 L.variegatus ops6 999 464 H.glaberrima ops6.2 Peropsins P.parvimensis ops6 H.glaberrima ops6.1 415 999 P.miniata ops6.2 A.rubens ops6 46 O.aranea ops6 Harrimaniidae sp ops7 995 S.mereschkowskii ops7 999 791 S.kowalevskii ops7 Schizocardium sp ops7 169 1000 P.flava ops7 429 798 P.bahamensis ops7 782 Torquaratorid sp ops7 617 B. aurantiacus ops7 Homo RGRopsin 686 H.glaberrima ops7 1000 S.purpuratus ops7 RGR-opsins 946 645 P.lividus ops7 998 O.aranea ops7 827 A.filiformis ops7 774 Amphipholis sp ops7 957 A.rubens ops7 P.miniata ops7.1 1000 P.miniata ops7.2 792 Ambulacraria Melatonin Receptors (Outgroup)

0.6 Figure 3 ECHINOPSINS A

Go OPSINS CILIARY OPSINS

ECHINOPSINS B

RHABDOMERIC OPSINS

NEUROPSINS

Go-OPSINS

PEROPSINS

RGR OPSINS

OUTGROUP (Melatonin Receptors) Figure 4

Hemichorda Echinodermata Phylum Torquaratoridae Holothuroidea Harrimaniidae Ptychoderidae Ophiuroidea Spengelidae Echinoidea Asteroidea Crinoidea Class Strongylocentrotus-droebachiensis Strongylocentrotus-intermedius Strongylocentrotus-purpuratus Balanoglossus-c.f.-aurantiacus Saccoglossus-mereschkowskii Schizocardium-c.f.-braziliense Hemicentrotus-pulcherrimus Heliocidaris-erythrogramma Parastichopus-parvamensis Harrimaniidae-sp.-(Iceland) Parastichopus-californicus Saccoglossus-kowalevskii Torquaratoid-sp.-(iceland) Florometra-serratissima Ptychodera-bahamensis Antedon-mediterranea Holothuria-glaberrima Lytechinus-variegatus Labidiaster-annulatus Astrotomma-agassizii Paracentrotus-lividus Eucidaris-tribuloides Amphiura-filiformis Leptosynapta-clarki Holothuria-forskali Ophiopsila-aranea Ptychodera-flava Patiria-miniata Asterias-rubens Amphipholis-sp Species 10 13 1 3 5 1 7 6 2 1 8 1 3 1 4 2 1 1 7 1 1 4 6 1 1 3 4 2 1 5 TOTAL 1 1 1 1 1 1 1 1 1 6 1 1 1 1 1 4 R0opsins3/3opsins34 1 1 1 1 1 2 2 1 1 1 1 1 1 1 1 C0opsins3/3opsins31 1 1 2 2 2 1 1 1 1 Go3opsins3/3opsins33 1 1 1 1 2 1 1 1 1 5 Neuropsins3/3opsins38 1 1 1 2 1 1 1 1 1 1 Peropsins3/3opsins36 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 RGR0opsins3/3opsins37 1 1 1 1 Echinopsins3A3/3opsins32 1 1 1 1 1 1 1 Echinopsins3B3/3opsins35 Supplementary Material File 1 Click here to download Supplementary Material: Suppl. File 1 NEW 29 September.xlsx

Supplementary Material File 2 Click here to download Supplementary Material: Suppl. File 2 NEW Transcriptome table.xlsx

Supplementary Material File 3 Click here to download Supplementary Material: Suppl. File 3 NEW Opsin Fasta.docx

Supplementary Material File 4 Click here to download Supplementary Material: Suppl. File 4 NEW Opsin alignment.phy.txt

Supplementary Material File 5 Click here to download Supplementary Material: Suppl. File 5 NEW Opsin alignment image 29Sept.pdf