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	Fig. 1. Phylogenetic analysis of bilaterian kisspeptin-type receptors, including A. rubens receptors ArKPR1-11. The phylogenetic tree, which was constructed using the maximum-likelihood method and rooted with galanin/allatostatin-A-type receptors as an outgroup, comprises three distinct clades, with bootstrap support > 90. Clade 1 includes ArKPR2-4 (ArubKPR2-4) and chordate kisspeptin-type receptors. Clade 2 includes ArKPR1 and protostome (annelid, mollusk) kisspeptin-type receptors. Clade 3 comprises three sub-clades: clade 3.1 includes ArKPR8-9 (ArubKPR8-9) and related receptors in other echinoderms and the hemichordate S. kowalevskii, clade 3.2 includes ArKPR5-7 (ArubKPR5-7) and related receptors in other echinoderms and the hemichordate S. kowalevskii, and clade 3.3 includes ArKPR10-11 (ArubKPR10-11) and related receptors in other echinoderms. The stars represent branch support (bootstrap 1000 replicates) and the pastel-colored backgrounds represent taxonomic groups (see key). The arrowheads label the eleven A. rubens kisspeptin-type receptors ArKPR1-11. The scale bar indicates amino acid substitutions per site. Receptors for which ligands have been identified experimentally in this study or other published studies are colored in blue. Species names are as follows: Apla, Acanthaster planci; Ajap, Apostichopus japonicus; Arub, Asterias rubens, Anjap Annessia japonica; Bbel, Branchiostoma belcheri; Ctel, Capitella teleta; Cgig, Crassostrea gigas; Hsap, Homo sapiens; Lcha, Latimeria chalumnae; Locu, Lepisosteus oculatus; Lgig, Lottia gigantea; Mmus, Mus musculus; Pdum, Platynereis dumerilii; Pbiv, Python bivittatus; Skow, Saccoglossus kowalevskii; Spur, Strongylocentrotus purpuratus; Xtro, Xenopus tropicalis. Accession numbers for the sequences of the receptors included in this tree are listed in Additional file 3
	Fig. 2. Comparative analysis of the structure and chromosomal location of genes encoding kisspeptin-type receptors. A Comparison of the exon/intron structure of genes encoding kisspeptin-type receptors in A. rubens (Ar), Homo sapiens (Hs), Xenopus tropicalis (Xt), Lepisosteus oculatus (Lo), and Crassostrea gigas (Cg). Exons are shown as rectangles, with non-coding regions white and protein-coding regions black or colored (regions encoding predicted transmembrane domains 1–7 are shown in red, orange, yellow, green, blue, dark purple, and light purple, respectively). Introns are shown as lines, with length underneath. The two introns highlighted with a pink bracket are a conserved feature (see also Additional file 9). Accession numbers for the transcripts/genes represented in this figure are in Additional file 2. B Diagram showing the chromosomal locations of genes encoding ArKPR1-11 in A. rubens. Genes encoding (i) ArKPR1-4, (ii) ArKPR5-9; ArKPR11, and (iii) ArKPR10 are located on chromosomes 15, 10, and 4, respectively. Curved lines linking genes correspond to clades in Fig. 1: light purple is clade 1, green is clade 3.1, orange is clade 3.2, and black is clade 3.3. C Diagram showing the relative locations and orientations of genes encoding (i) ArKPR1-4 (clade 1 and 2 of Fig. 1) in a region of chromosome 15, (ii) ArKPR5-7 (clade 3.2 of Fig. 1) in a region of chromosome 10, (iii) ArKPR8-9 (clade 3.1 and 3.3 of Fig. 1) in a region of chromosome 10, and (iv) ArKPR10 (clade 3.3 of Fig. 1). The length (including exons and introns) and orientation of the genes is indicated by the purple arrows and the distance between genes (number of bases) is stated underneath the intervening black lines. The absence (0 genes) or presence (number of genes) of other genes in between those encoding the kisspeptin-type receptors is also stated (see Additional file 11 for data)
	Fig. 3. Neuropeptides identified as candidate ligands for kisspeptin-type receptors in echinoderms aligned with chordate kisspeptins. A Sequences of the A. rubens kisspeptin-type precursors ArKPP1 and ArKPP2 and SALMFamide-type precursors ArL-SALMFaP and ArF-SALMFaP. N-terminal signal peptide is shown in blue, neuropeptides predicted or shown to be derived from these proteins are shown in red (with C-terminal glycine that is a substrate for amidation shown in orange) and monobasic or dibasic cleavage sites are shown in green. Neuropeptides (red) are named in accordance with the precursor they are derived from and their relative position in the precursor, which explains the nomenclature used in B–D. Likewise, annotated precursor sequences for other neuropeptides included in B and C are shown in Additional file 13. B Alignment of ArKP1.1, ArKP1.2, and KP1-type neuropeptides from other echinoderms (blue) with chordate kisspeptin-type neuropeptides (yellow). C Alignment ArKP2.1, ArKP2.2, and KP2-type neuropeptides from other echinoderms (blue) with chordate kisspeptin-type neuropeptides (yellow). D Alignment of L-type SALMFamide precursor-derived peptides from A. rubens (ArS1.1, ArS1.3, ArS1.4, and S1.7) and other echinoderms (blue) with chordate kisspeptin-type neuropeptides (yellow). E Alignment of F-type SALMFamide precursor-derived peptides from A. rubens (ArS2.2, ArS2.3, and S2.8) and other echinoderms (blue) with chordate kisspeptin-type neuropeptides (yellow). Conserved residues are highlighted in black or gray. Abbreviations: Ar, Asterias rubens; Sp, Strongylocentrotus purpuratus; Aj, Apostichopus japonicus, Hs, Homo sapiens, Lo, Lepisosteus oculatus, Bf, Branchiostoma floridae. The accession numbers for the precursor sequences used for this figure are in Additional file 13
	Fig. 4. Structure and chromosomal location of genes encoding candidate ligands for A. rubens kisspeptin-type receptors. A Comparison of the exon/intron structure of genes encoding precursors of candidate ligands for kisspeptin-type receptors in A. rubens (Ar) and genes encoding kisspeptin-type precursors in Homo sapiens (Hs), Xenopus tropicalis (Xt), and Lepisosteus oculatus (Lo). Exons are shown as rectangles, with non-coding regions white and protein-coding regions black or colored (regions encoding the N-terminal signal peptide, neuropeptides and predicted monobasic/dibasic cleavage sites are shown in blue, red, and green, respectively). Introns are shown as lines, with length underneath. The presence of a phase 1 intron interrupting the coding sequence between exons encoding the N-terminal signal peptide and one or more neuropeptides is a feature that is conserved between the A. rubens KPP1 gene and vertebrate kisspeptin precursor genes, providing evidence of orthology, whereas ArKPP2 is encoded by a single exon. The presence of an intron that interrupts the coding sequence between an exon encoding the N-terminal signal peptide and an exon encoding multiple neuropeptides is also a feature of the two A. rubens SALMFamide precursor genes, but this is a phase 0 intron. The presence and position of introns shown here in A. rubens neuropeptide precursor genes are conserved in orthologs in other echinoderm species (see Additional file 17). The accession numbers for the sequences of the precursors shown in this figure are listed in Additional file 17. B Diagram showing the chromosomal locations of four genes encoding precursors of candidate ligands for kisspeptin-type receptors in A. rubens: ArKPP1, ArKPP2, ArL-SALMFaP, and ArF-SALMFaP. Genes encoding ArKPP1 and ArKPP2 are located on chromosome 22 and genes encoding ArL-SALMFaP and ArF-SALMFaP are located on chromosome 13, indicating that these are two pairs of paralogous genes that originated by gene duplication and intrachromosomal translocation
	Fig. 5. Identification of neuropeptides that act as ligands for A. rubens kisspeptin-type receptors. A ArKPR1 is only activated by ArKP1.2 (EC50 = 1.16 × 10−8 M). B ArKPR3 is only activated by ArKP2.2 (EC50 = 5.80 × 10−10 M). C ArKPR6 is activated by multiple neuropeptides at high concentrations (> 1 µM) but is only activated by a ‘cocktail’ of neuropeptides derived from the F-type SALMFamide precursor at lower concentrations (EC50 = 1.33 × 10−9 M). D ArKPR7 is activated by multiple neuropeptides at high concentrations (> 1 µM) but is only activated by a ‘cocktail’ of neuropeptides derived from the L-type SALMFamide precursor at lower concentrations (EC50 = 2.43 × 10−9 M). E ArKPR8 is activated by both ArKP1.1 and ArKP1.2 at high concentrations (> 1 µM) but is only activated by ArKP1.1 at lower concentrations (EC50 = 3.88 × 10−7 M). F ArKPR9 is activated by ArKP1.1, ArKP1.2, and a ‘cocktail’ of neuropeptides derived from the L-type SALMFamide precursor, but ArKP1.2 is the most potent ligand for this receptor (EC50 = 3.07 × 10−.8 M). Key: green = ArKP1.1; pink = ArKP1.2; purple = ArKP2.2; blue = ‘cocktail’ of neuropeptides derived from the L-type SALMFamide precursor (ArS1.1–7); red = ‘cocktail’ of neuropeptides derived from the F-type SALMFamide precursor (ArS2.1–8). Each point represents mean values (± S.E.M.) from at least four independent experiments, with each experiment performed in triplicate. Luminescence is expressed as a percentage of the maximal response observed in each experiment. The source data for these experiments is provided in Additional file 15

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