Semmens DC , Mirabeau O , Moghul I , Pancholi MR , Wurm Y , Elphick MR .

	Figure 1. Animal phylogeny. Phylogenetic diagram showing the position of the phylum Echinodermata (shown in red; e.g. starfish) in the deuterostomian branch of the animal kingdom. The Bilateria comprise two super-phyla—the deuterostomes and the protostomes. The deuterostomes comprise the chordates (vertebrates, urochordates and cephalochordates) and the ambulacrarians (hemichordates and echinoderms). The protostomes comprise the lophotrochozoans (e.g. molluscs and annelids) and the ecdysozoans (e.g. arthropods and nematodes). The Cnidaria (e.g. sea anemones) are basal to the Bilateria. Images of representative animals from each phylum were obtained from http://phylopic.org or were created by the authors or by M. Zandawala (Stockholm University).
	Figure 2. Precursors of neuropeptides in A. rubens that provide novel insights into neuropeptide evolution at the superphylum/phylum level. Predicted signal peptides are shown in blue, putative neuropeptides are shown in red (with cysteine (C) residues underlined), C-terminal glycine (G) residues that are putative substrates for amidation are shown in orange and putative dibasic/tribasic cleavage sites are shown in green.
	Figure 3. Alignment of ArKP1 and ArKP2 with other kisspeptin (KP)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens KP-type precursor [GenBank: KT601705]; Spur, S. purpuratus KP-type precursor [GI:374768013]; Skow, S. kowalevskii KP-type precursor [GI:187123982]; Bflo1, B. floridae KP-type precursor 1 [GI:260826607]; Bflo2, B. floridae KP-type precursor 2 [GI:260827077]; Bflo3, B. floridae KP-type precursor 3 [GI:260826605]; Bflo4, B. floridae KP-type precursor 4 [GI:260793233]; Hsap, Homo sapiens KiSS-1 metastasis-suppressor precursor [GI:21950713].
	Figure 4. Alignment of ArMCH with other melanin-concentration hormone (MCH)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens MCH-type precursor [GenBank: KT601706]; Spur, S. purpuratus MCH-type precursor [GI:109402760]; Skow, S. kowalevskii MCH-type precursor [GI:187231810]; Trub, Takifugu rubripes MCH precursor [GI:410918650]; Hsap, H. sapiens MCH precursor [GI:187445].
	Figure 5. Alignment of ArTK1 and ArTK2 with other tachykinin (TK)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens TK-type precursor [GenBank: KT601707]; Spur, S. purpuratus TK-type precursor [GI:109402899]; Cint, C. intestinalis TK-type precursor [GI:74136064]; Hsap_SubP, H. sapiens β-prepro TK precursor [GI:29482]; Hsap_NKB, H. sapiens neurokinin-β precursor [GI:48146502]; Hsap_NKA, H. sapiens TK4 precursor [GI:117938255]; Ctel, Capitella teleta [GI:161289578]; Lgig1, Lottia gigantea TK-type precursor 1 [GI:676441944]; Lgig2, L. gigantea TK-type precursor 2 [GI:163525452]; Dmel, D. melanogaster TK precursor [GI:442618676].
	Figure 6. Alignment of ArSS with other somatostatin (SS)-type peptides and AST-C-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens SS-type precursor [GenBank: KT601708]; Spur, S. purpuratus SS-type precursor [GI:390344260]; Bflo, B. floridae SS-type precursor [JGI:72051]; Hsap_SS, H. sapiens SS precursor [GI:21619155]; Hsap_CORT, H. sapiens cortistatin (CORT) precursor [GI:110645815]; Ctel, C. teleta AST-C-type precursor [GI:161295377]; Lgig, L. gigantea AST-C-type precursor [GI:163505903]; Dmel_AstC, D. melanogaster AST-C-type precursor [GI:665407583]; Dmel_AstCC, D. melanogaster AST-CC-type precursor [GI:665407585].
	Figure 7. Alignment of ArPDF1 and ArPDF2 with other pigment-dispersing factor (PDF)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens PDF-type precursor [GenBank: KT601709]; Spur, S. purpuratus PDF-type precursor [GI:115899431]; Skow, S. kowalevskii PDF-type precursor [GI:187067819]; Ctel, C. teleta PDF-type precursor [JGI:204689]; Lgig, L. gigantea cerebrin precursor [GI:676458325]; Dmel, D. melanogaster PDF precursor [GI:281362639]; Cele, C. elegans PDF precursor [GI:25149644].
	Figure 8. Alignment of ArCRH with other corticotropin-releasing hormone (CRH)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens CRH-type precursor [GenBank: KT601710]; Skow1, S. kowalevskii CRH-type precursor 1 [GI:281433636]; Skow2, S. kowalevskii CRH-type precursor 2 [GI:281433636]; Bflo, B. floridae CRH-type precursor [GI:260786674]; Hsap_CRH, H. sapiens CRH precursor [GI:30583744]; Hsap_UCN, H. sapiens urocortin (UCN) precursor [GI:49457481]; Hsap_UCN2, H. sapiens stresscopin-related protein precursor [GI:14029393]; Hsap_UCN3, H. sapiens stresscopin precursor [GI:15026913]; Ctel1, C. teleta CRH-type precursor 1 [GI:161303031]; Ctel2, C. teleta CRH-type precursor 2 [JGI:190906]; Ctel3, C. teleta CRH-type precursor 3 [JGI:190906]; Ctel4, C. teleta CRH-type precursor 4 [JGI:194553]; Lgig1, L. gigantea CRH-type precursor 1 [GI:676493124]; Lgig2, L. gigantea CRH-type precursor 2 [GI:163524672].
	Figure 9. Precursors of neuropeptides in A. rubens that are novel echinoderm representatives of bilaterian neuropeptide families. Predicted signal peptides are shown in blue, putative neuropeptides are shown in red (with cysteine (C) residues underlined), C-terminal glycine (G) residues that are putative substrates for amidation are shown in orange and putative dibasic cleavage sites are shown in green. For the (a) asterotocin and (b) NGFFYamide precursors, the C-terminal neurophysin domain (with the characteristic 14 cysteine (c) residues underlined) is shown in purple.
	Figure 10. Alignment of asterotocin with other vasopressin/oxytocin (VP/OT)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens asterotocin precursor [GenBank: KT601711], Spur, S. purpuratus echinotocin precursor [GI:390337108]; Skow, S. kowalevskii VP/OT-type precursor [GI:187155721]; Bflo, B. floridae VP/OT-type precursor [GI:260828088]; Hsap_VP, H. sapiens VP precursor [GI:340298]; Hsap_OT, H. sapiens OT precursor [GI:189410]; Ctel, C. teleta VP/OT-type precursor [JGI:173251]; Lgig, L. gigantea VP/OT-type precursor [JGI:53893]; Dpul, Daphnia pulex VP/OT-type precursor [JGI:59567].
	Figure 11. Alignment of A. rubens GnRH-type peptides/precursors with other gonadotropin-releasing hormone (GnRH)-type peptides/precursors. (a) Alignment of GnRH-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens GnRH-type precursor 1 [GenBank: KT601712]; Spur, S. purpuratus GnRH-type precursor 1 [GI:390361802]; Skow, S. kowalevskii GnRH-type precursor [GI:585702722]; Bflo, B. floridae GnRH-type precursor [GI:568818760]; Hsap1, H. sapiens GnRH precursor 1 [GI:133908609]; Hsap2, H. sapiens GnRH precursor 2 [GI:109731929]; Ctel, C. teleta GnRH-type precursor [GI:161294493]; Acal, A. californica GnRH-type precursor [GI:325296898]; Dmel_CORZ, D. melanogaster corazonin (CORZ) precursor [GI:386765761]; Dmel_AKH, D. melanogaster adipokinetic hormone (AKH) precursor [GI:281365621]. (b) Alignment of GnRH-type precursors. Accession numbers for the corresponding precursor proteins are: Arub_GnRH1P, A. rubens GnRH-type precursor 1 [GenBank: KT601712]; Spur_GnRH1P, S. purpuratus GnRH-type precursor 1 [GI:390361802]; Arub_GnRH2P, A. rubens GnRH-type precursor 2 [GenBank: KT601713]; Spur_GnRH2P, S. purpuratus GnRH-type precursor 2 [GI:109403263].
	Figure 12. Alignment of ArTRH with other thyrotropin-releasing hormone (TRH)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens TRH-type precursor [GenBank: KT601714]; Spur, S. purpuratus TRH-type precursor [GI:109402869]; Skow, S. kowalevskii TRH-type precursor [GI:187216047]; Bflo, B. floridae TRH-type precursor [GI:260784028]; Hsap, H. sapiens TRH precursor [GI:485464565]; Pdum, P. dumerilii EFLGamide precursor [GI:332167915].
	Figure 13. Alignment of ArCT with other calcitonin (CT)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens CT-type precursor [GenBank: KT601715]; Spur, S. purpuratus CT-type precursor [GI:115767208]; Skow, S. kowalevskii CT-type precursor [GI:187217193]; Bflo1, B. floridae CT-type precursor 1 [GI:260826569]; Bflo2, B. floridae CT-type precursor 2 [GI:260826567]; Bflo3, B. floridae CT-type precursor 3 [GI:260826573]; Bflo4, B. floridae CT-type precursor 4 [GI:260812099]; Hsap_Calc, H. sapiens CT precursor [GI:179819]; Hsap_CGRP1, H. sapiens CT gene-related peptide (CGRP) 1 precursor [GI:269784661]; Hsap_IAPP, H. sapiens islet amyloid polypeptide (IAPP) precursor [GI:109255169]; Hsap_ADML, H. sapiens adrenomedullin precursor [GI:675022745]; Hsap_ADM2, H. sapiens adrenomedullin 2 precursor [GI:41016725]; Ctel, C. teleta CT-type precursor [GI: 161220966]; Lgig1, L. gigantea CT-type precursor 1 [GI:163526287]; Lgig2, L. gigantea CT-type precursor 2 [GI:676481265].
	Figure 14. Alignment of ArCCK1 and ArCCK2 with other cholecystokinin (CCK)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens CCK-type precursor [GenBank: KT601716]; Spur, S. purpuratus CCK-type precursor [GI:390355380]; Skow1, S. kowalevskii CCK-type precursor 1 [GI:585688033]; Skow2, S. kowalevskii CCK-type precursor 2 [GI:187061456]; Cint, C. intestinalis cionin precursor [GI:10799472]; Hsap_CCK-8, H. sapiens CCK precursor [GI:30582820]; Hsap_Gast-6, H. sapiens gastrin precursor [GI:47481291]; Ctel, C. teleta CCK-type precursor [GI:161296032]; Lgig1, L. gigantea CCK-type precursor 1 [GI:161296032]; Lgig2, L. gigantea CCK-type precursor 2 [GI:52414496]; Dmel_SK, D. melanogaster sulfakinin (SK) precursor [GI:386765036].
	Figure 15. Alignment of ArOX1 and ArOX2 with other orexin (OX)-type peptides. Accession numbers for the corresponding precursor proteins are: Arub1, A. rubens OX-type precursor 1 [GenBank: KT601717]; Arub2, A. rubens OX-type precursor 2 [GenBank: KT601718]; Spur1, S. purpuratus OX-type precursor 1 [GI:346420309]; Spur2, S. purpuratus OX-type precursor 2 [GI:346419879]; Skow, S. kowalevskii OX-type precursor [GI:585662697]; Bflo1, B. floridae OX-type precursor 1 [GI:260807454]; Bflo2, B. floridae OX-type precursor 2 [GI:260780718]; Hsap_OX, H. sapiens OX precursor [GI:4557634].
	Figure 16. Alignment of ArLQ with other luqin (LQ)-type peptides and alignment of a conserved C-terminal domain of LQ-type precursor proteins. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens LQ-type precursor [GenBank: KT601719]; Spur, S. purpuratus LQ-type precursor [GI:390331828]; Skow, S. kowalevskii LQ-type precursor [GI:585716464]; Ctel, C. teleta LQ-type precursor [GI:161280144]; Lgig, L. gigantea LQ-type precursor [GI:163510328]; Dpul, D. pulex LQ-type precursor [JGI:251691]; Dvir, Drosophila virilis LQ-type precursor [GI: 968114152].
	Figure 17. Alignment of representative A. rubens PP/orcokinin-type peptide with other PP/orcokinin-type peptides. Accession numbers for the corresponding precursor proteins are: Arub, A. rubens PP/orcokinin-type precursor [GenBank: KT601720]; Spur1, S. purpuratus PP/orcokinin-type precursor 1 [GI:390335272]; Spur2, S. purpuratus PP/orcokinin-type precursor 2 [GI:390352581]; Ctel, C. teleta PP-type precursor [GI:161190484]; Lgig, L. gigantea PP-type precursor [GI:163513756]; Dmel, D. melanogaster orcokinin precursor [GI:442624594].
	Figure 18. Precursors of cysteine-rich peptide hormones and growth factors in A. rubens. Predicted signal peptides are shown in blue, putative peptide hormones/growth-factors are shown in red (with cysteine (C) residues underlined), C-terminal glycine (G) residues that are putative substrates for amidation are shown in orange and putative dibasic cleavage sites are shown in green. For ArIGFP1 and ArIGFP2, the C-peptide is shown in black, the D-domain (for ArIGFP1) is shown in olive and the E-domain is shown in maroon.
	Figure 19. Alignment of glycoprotein/bursicon hormone-type precursors. Accession numbers for the corresponding precursor proteins are: Arub_GPA2_1, A. rubens glycoprotein hormone α-2 (GPA2)-type precursor 1 [GenBank: KT601721]; Arub_GPA2_2, A. rubens GPA2-type precursor 2 [GenBank: KT601722]; Arub_GPB5_1, A. rubens glycoprotein hormone β-5 (GPB5)-type precursor 1 [GenBank: KT601723], Arub_GPB5_2, A. rubens GPB5-type precursor 2 [GenBank: KT601724]; Arub_GPB5_3, A. rubens GPB5-type precursor 3 [GenBank: KT601725]; Arub_BursA, A. rubens bursicon-α-type precursor [GenBank: KT601726]; Arub_BursB, A. rubens bursicon-β-type precursor [GenBank: KT601727]; Hsap_GPA2, H. sapiens GPA2 precursor [GI:189491650]; Hsap_GPB5, H. sapiens GPB5 precursor [GI:21427593]; Hsap_FSHB, H. sapiens follicle-stimulating hormone (FSH) β-precursor [GI:124014246]; Hsap_CGB7, H. sapiens chorionic gonadotropin (CG) β-polypeptide 7 precursor [GI:15451749]; Hsap_LSHB, H. sapiens luteinizing hormone (LH) β-polypeptide precursor [GI:15431286]; Hsap_TSHB, H. sapiens the putative IGF-type receptor (TSH) β-precursor [GI:47479817]; Dmel_GPA2, D. melanogaster GPA2 precursor [GI:320546230]; Dmel_TSHB, D. melanogaster glycoprotein hormone β-subunit-related protein precursor [GI:21427595]; Dmel_BursA, D. melanogaster bursicon-α precursor [GI:665394724]; Dmel_BursB, D. melanogaster bursicon-β precursor [GI:62392020].
	Figure 20. Alignment of insulin/relaxin/insulin-like growth factor (IGF)-type precursors. Accession numbers for the corresponding precursor proteins are: Arub_Rel1, A. rubens relaxin-like gonad-stimulating peptide precursor [GenBank: KT601728]; Arub_Rel2, A. rubens relaxin-like peptide precursor 2 [GenBank: KT601729]; Arub_IGF1, A. rubens IGF-type precursor 1 [GenBank: KT601730]; Arub_IGF2, A. rubens IGF-type precursor 2 [GenBank: KT601731]; Spur_IGF1, S. purpuratus IGF-type precursor 1 [GI:390333650]; Spur_IGF2, S. purpuratus IGF-type precursor 2 [GI:390333648]; Skow, S. kowalevskii IGF-type precursor [GI:187064073]; Bflo1, B. floridae IGF-type precursor 1 [JGI:72897]; Bflo2, B. floridae IGF-type precursor 2 [JGI:74371]; Bflo3, B. floridae IGF-type precursor 3 [JGI:77763]; Bflo4, B. floridae IGF-type precursor 4 [JGI:100967]; Hsap_INS, H. sapiens insulin (INS) precursor [GI:186429]; Hsap_IGF1, H. sapiens IGF precursor [GI:49456676]; Hsap_REL1, H. sapiens relaxin-1 precursor [GI:35932]; Hsap_REL2, H. sapiens relaxin-2 precursor [GI:35926]; Hsap_REL3, H. sapiens relaxin-3 precursor [GI:17484095]; Dmel_INSL1, D. melanogaster insulin-like (INSL) peptide 1 precursor [GI:317423340]; Dmel_INSL2, D. melanogaster INSL peptide 2 precursor [GI:442631434]; Dmel_INSL3, D. melanogaster INSL peptide 3 precursor [GI:221331056]; Dmel_INSL4, D. melanogaster INSL peptide 4 precursor [GI:442631435]; Dmel_INSL5, D. melanogaster INSL peptide 5 precursor [GI:320545737]; Dmel_INSL6, D. melanogaster INSL peptide 6 precursor [GI:442614930]; Dmel_INSL7, D. melanogaster INSL peptide 7 precursor [GI:386763756]; Dmel_INSL8, D. melanogaster INSL peptide 8 precursor [GI:386771312].
	Figure 21. Precursors of putative neuropeptides in A. rubens that do not share apparent sequence similarity with known neuropeptide families. Predicted signal peptides are shown in blue, putative peptides are shown in red (with cysteine (C) residues underlined), C-terminal glycine (G) residues that are putative substrates for amidation are shown in orange and putative dibasic cleavage sites are shown in green.

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