Zandawala M , Moghul I , Yañez Guerra LA , Delroisse J , Abylkassimova N , Hugall AF , O'Hara TD , Elphick MR .

BB/M001644/1 Biotechnology and Biological Sciences Research Council , BB/M009513/1 Biotechnology and Biological Sciences Research Council

	Figure 1. Bilaterian animal phylogeny. The diagram shows (i) the phylogenetic position of the phylum Echinodermata in the ambulacrarian clade of the deuterostomes and (ii) relationships between the five extant classes of echinoderms, which include the focal class for this study—the Ophiuroidea (e.g. Ophionotus victoriae).
	Figure 2. Eclosion hormone (EH)-type peptides and receptors in echinoderms. (a) Partial multiple sequence alignment of EH-type precursor sequences, excluding the N-terminal signal peptide. (b) Cluster analysis of arthropod EH precursors, echinoderm EH-like precursors, arthropod ion transport peptides (ITPs) and vertebrate atrial natriuretic peptides (ATPs) shows that echinoderm EH-like precursors are more closely related to arthropod EH than ITP. (c) Maximum-likelihood and Bayesian phylogenetic analyses of membrane guanylate cyclase receptors show that EH-like receptors are found in echinoderms, but are absent in vertebrates as seen for the EH-like precursors. OGC1, 2, 3 and 4 are orphan guanylate cyclase receptors found in arthropods [33]. Echinoderm EH-like receptors are clustered with arthropod EH receptors, neuropeptide-like peptide 1-VQQ receptors (NPLP1-VQQ) and OGC1 receptors. The inset shows the alternate topology obtained following Bayesian analysis. Species names: Ophionotus victoriae (Ovic), Asterias rubens (Arub), Strongylocentrotus purpuratus (Spur), Drosophila melanogaster (Dmel), Bombyx mori (Bmor) and Pediculus humanus corporis (Pcor).
	Figure 3. Multiple sequence alignments of (a) CCHamide-type and (b) neuropeptide-F/Y (NPF/NPY) type peptides. Species names: Ophionotus victoriae (Ovic), Asterias rubens (Arub), Apostichopus japonicus (Ajap), Drosophila melanogaster (Dmel), Apis mellifera (Amel), Lottia gigantea (Lgig), Aplysia californica (Acal), Homo sapiens (Hsap), Ophiopsila aranea (Oara), Amphiura filiformis (Afil), Patiria miniata (Pmin), Saccoglossus kowalevskii (Skow), Branchiostoma floridae (Bflo) and Daphnia pulex (Dpul).
	Figure 4. Summary of neuropeptide precursors identified in Ophionotus victoriae, Amphiura filiformis and Ophiopsila aranea. Neuropeptide precursors are classified based on the type of G-protein coupled receptor (GPCR) their constituent peptides are predicted to activate (see Mirabeau and Joly [6]). Some peptides bind to receptors other than GPCRs and these are grouped with peptides where the receptor is unknown. Ophiuroids have neuropeptide precursors from up to 32 families. The number of putative mature peptides derived from each precursor has been indicated along with the presence of amidation and pyroglutamation.
	Figure 5. Multiple sequence alignments of mature peptides belonging to selected neuropeptide families. (a) Corazonin alignment; (b) gonadotropin-releasing hormone (GnRH) alignment; (c) orexin alignment; (d) luqin alignment; (e) vasopressin/oxytocin (VP/OT) alignment; (f) Ovnp18 alignment; (g) MCH alignment; (h) NG peptide alignment; (i) pigment-dispersing factor (PDF) alignment (see electronic supplementary material, figure S10 for a multiple sequence alignment of PDF-type precursors). Species names: Ophionotus victoriae (Ovic), Asterias rubens (Arub), Strongylocentrotus purpuratus (Spur), Apostichopus japonicus (Ajap), Saccoglossus kowalevskii (Skow), Branchiostoma floridae (Bflo), Anopheles gambiae (Agam), Daphnia pulex (Dpul), Strigamia maritima (Smar), Lottia gigantea (Lgig) and Homo sapiens (Hsap).
	Figure 6. Alignments of neuropeptides derived from precursors that exist in multiple forms in ophiuroids. (a) Thyrotropin-releasing hormone (TRH) alignment; (b) Cholecystokinin (CCK) alignment; (c) somatostatin alignment; (d) Corticotropin-releasing hormone (CRH) alignment. Species names: Ophionotus victoriae (Ovic), Asterias rubens (Arub), Strongylocentrotus purpuratus (Spur), Apostichopus japonicus (Ajap), Branchiostoma floridae (Bflo), Homo sapiens (Hsap), Drosophila melanogaster (Dmel) and Lottia gigantea (Lgig).
	Figure 7. Comparative analysis of ophiuroid tachykinin, KP and calcitonin-type precursors and neuropeptides. (a) Alignment of tachykinin-type peptides in O. victoriae (Ophiuroidea) and A. rubens (Asteroidea). (b) Schematic diagrams of the O. victoriae and A. rubens tachykinin precursors showing the location of the signal peptide (SP) and predicted neuropeptides (labelled 1–4). (c) Alignments of the long and short forms of kisspeptin (KP)-type neuropeptides in O. victoriae, A. rubens and S. purpuratus (Echinoidea). (d) Schematic diagrams of the O. victoriae and A. rubens KP precursors showing the locations of the SP, short and long orthocopies and cysteine (C) residues. (e) Alignment of calcitonin-type peptides from O. victoriae, A. rubens, S. purpuratus and A. japonicus (Holothuroidea). (f) Predicted alternative splicing of the calcitonin gene in ophiuroids, with the location of the SP and neuropeptides (CT1 and CT2) labelled. Species names: Ophionotus victoriae (Ovic), Asterias rubens (Arub), Strongylocentrotus purpuratus (Spur) and Apostichopus japonicus (Ajap).
	Figure 8. Comparison of neuropeptide copy numbers across the Ophiuroidea for precursors comprising multiple copies of neuropeptides. Neuropeptide precursors were mined from 52 ophiuroid transcriptomes, with the phylogeny adapted from O'Hara et al. [12]. Am_laud: Amphiophiura laudata, Am_spat: Amphiophiura spatulifera, Am_cipu: Amphioplus cipus, Am_cten: Amphioplus ctenacantha, Am_squa: Amphipholis squamata, Am_cons1: Amphiura constricta 1, Am_cons2: Amphiura constricta 2, As_love: Asteronyx loveni, As_bidw: Asteroschema bidwillae, As_tubi: Asteroschema tubiferum, Ba_hero: Bathypectinura heros, Cl_cana: Clarkcoma canaliculata, Gl_sp_no: Glaciacantha sp. nov., Go_pust: Gorgonocephalus pustulatum, Mi_grac: Microphiopholis gracillima, Op_fune: Ophiacantha funebris, Op_abys: Ophiactis abyssicola, Op_resi: Ophiactis resiliens, Op_savi: Ophiactis savignyi, Op_vall: Ophiernus vallincola, Op_pilo: Ophiocentrus pilosus, Op_wend: Ophiocoma wendtii, Op_oedi: Ophiocreas oedipus, Op_tube: Ophiocypris tuberculosis, Op_appr: Ophioderma appressum, Op_bisc: Ophiolepis biscalata, Op_impr: Ophiolepis impressa, Op_brev: Ophioleuce brevispinum, Op_perf: Ophiolimna perfida, Op_prol: Ophiologimus prolifer, Op_obst: Ophiomoeris obstricta, Op_lyma: Ophiomusium lymani, Op_aust: Ophiomyxa australis, Op_vivi: Ophiomyxa sp. cf. vivipara, Op_fasc: Ophionereis fasciata, Op_reti: Ophionereis reticulata, Op_scha: Ophionereis schayeri, Op_cyli: Ophiopeza cylindrica, Op_filo: Ophiophragmus filograneus, Op_wurd: Ophiophragmus wurdemanii, Op_liod: Ophiophrura liodisca, Op_john: Ophiophycis johni, Op_lame: Ophioplax lamellosa, Op_iner: Ophiopleura inermis, Op_plic: Ophioplinthaca plicata, Op_bisp: Ophioplocus bispinosus, Op_macu: Ophiopsammus maculata, Op_angu: Ophiothrix angulata, Op_caes: Ophiothrix caespitosa, Op_exim_1: Ophiotreta eximia 1, Op_exim_2: Ophiotreta eximia 2, Op_sp_no: Ophiura sp. nov.
	Figure 9. A partial multiple sequence alignment of ophiuroid TRH-1 precursors showing clade-specific gain/loss of neuropeptide copies. Mono- and dibasic cleavage sites are highlighted in green, mature peptides in red with the glycine residue for amidation in pink. Species have been grouped and coloured (clade A in purple, clade B in blue and clade C in orange) based on the phylogeny determined by O'Hara et al. [12].
	Figure 10. A partial multiple sequence alignment of ophiuroid F-type SALMFamide precursors showing clade-specific gain/loss of neuropeptide copies. Dibasic cleavage sites are highlighted in green, and mature peptides in red with the glycine residue for amidation in pink. Species have been grouped and coloured (clade A in purple, clade B in blue and clade C in orange) based on the phylogeny determined by O'Hara et al. [12].

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