Phylogenetic analysis of SALMFamide precursors in echinoderms.

<p><b>A.</b> Echinoderm phylogeny. The basal position of crinoids and the sister group status of echinoids and holothurians is widely accepted but there is conflicting evidence with respect to the phylogenetic position of asteroids and ophiuroids <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0059076#pone.0059076-Pisani1" target="_blank">[24]</a>; therefore the three possible echinoderm phylogenies are shown in Ai, Aii and Aiii. <b>B.</b> Phylogenetic diagram showing the occurrence and organisation of SALMFamide precursors in species representing three echinoderm classes: the Echinoidea (<i>Strongylocentrotus purpuratus</i>; Sp), the Holothuroidea (<i>Apostichopus japonicus</i>; Aj) and the Asteroidea (<i>Patiria miniata</i>; Pm). Signal peptides are shown in blue and dibasic or monobasic cleavage sites are shown in green. L-type SALMFamides with the canonical C-terminal LxFamide motif are shown in red and peptides that are “L-type-like” (e.g. MGFTGNTGILLamide in <i>A. japonicus</i>, where the L and F are replaced by I and L, respectively) are shown in red with hatched shading. Likewise, F-type SALMFamides with the canonical C-terminal FxFamide motif are shown in yellow and peptides that are “F-type-like” (e.g. ADLFRSYAFamide in <i>P. miniata</i>, where one of the F residues is replaced by Y) are shown in yellow with hatched shading. In each species (class) there are two types of SALMFamide precursor: Firstly, a precursor that is exclusively comprised of L-type peptides or L-type-like peptides. Secondly, a precursor that is either exclusively comprised of F-type peptides (Sp) or a precursor that is largely comprised of F-type peptides or F-type-like peptides together with one or more L-type or L-type-like peptides (Aj and Pm).</p

<i>Patiria miniata</i> L-type SALMFamide precursor.

<p>The sequence of a gene (lowercase) in <i>P. miniata</i> that encodes an L-type SALMFamide precursor protein (bold uppercase, 174 amino acid residues) is shown. The majority of a large intron that separates the two exons is not shown (dashed line) but the 5′ and 3′ regions are shown, including the respective splice sites gt and ag, which are highlighted in bold. The predicted signal peptide of the precursor protein is shown in blue and the seven putative SALMFamide neuropeptides are shown in red flanked by putative dibasic cleavage sites (KR or RR) shown in green. The asterisk shows the position of the stop codon.</p

<i>Patiria miniata</i> F-type SALMFamide precursor.

<p>The sequence of a gene (lowercase) in <i>P. miniata</i> that encodes an F-type SALMFamide precursor protein (bold uppercase, 258 amino acid residues) is shown. The majority of a large intron that separates the two exons is not shown (dashed line) but the 5′ and 3′ regions are shown, including the respective splice sites gt and ag, which are highlighted in bold. The predicted signal peptide of the precursor protein is shown in blue and the nine putative SALMFamide neuropeptides are shown in red flanked by putative dibasic cleavage sites (KR or RR) shown in green. The asterisk shows the position of the stop codon.</p

Comparative analysis of L-type SALMFamide precursors and putative SALMFamides derived from L-type SALMFamide precursors.

<p><b>A.</b> Multiple sequence alignment of L-type SALMFamide precursors from <i>Strongylocentrotus purpuratus</i> (Sp), <i>Apostichopus japonicus</i> (Aj) and <i>Patiria miniata</i> (Pm). The symbol * labels the positions of residues that are identical in all three sequences, whilst the symbols : and. label the positions of strongly and weakly conserved residues, respectively. Putative neuropeptides that are aligned in all three sequences or that are aligned in the Pm sequence and the Sp or Aj sequences are labelled: Align L1, Align L2 and Align L3. <b>B.</b> Comparison of the C-terminally aligned sequences of putative SALMFamides derived from the L-type SALMFamide precursors in Sp, Aj and Pm. Hydrophobic residues are shown in red, hydrophilic residues are shown in green and basic residues are shown in pink. The putative C-terminal amide group is denoted as “a”.</p

<i>Apostichopus japonicus</i> L-type SALMFamide precursor.

<p>The cDNA sequence (lowercase, 3072 bases) of isotig 08429, which encodes an L-type SALMFamide precursor protein (bold uppercase, 176 amino acid residues) is shown. The predicted signal peptide is shown in blue and the three putative SALMFamide neuropeptides are shown in red flanked by putative dibasic cleavage sites (KR or KK) shown in green. The asterisk shows the position of the stop codon.</p

Comparative analysis of F-type SALMFamide precursors and putative SALMFamides derived from F-type SALMFamide precursors.

<p>A. Multiple sequence alignment of F-type SALMFamide precursors from <i>Strongylocentrotus purpuratus</i> (Sp), <i>Apostichopus japonicus</i> (Aj) and <i>Patiria miniata</i> (Pm). The symbol * labels the positions of residues that are identical in all three sequences, whilst the symbols : and. label the positions of strongly and weakly conserved residues, respectively. Putative neuropeptides that are aligned in all three sequences or that are aligned in the Pm sequence and the Sp or Aj sequences are labelled: Align F1– AlignF7. <b>B.</b> Comparison of the C-terminally aligned sequences of putative SALMFamides derived from the F-type SALMFamide precursors in Sp, Aj and Pm. Hydrophobic residues are shown in red, hydrophilic residues are shown in green, acidic residues are shown in blue and basic residues are shown in pink. The putative C-terminal amide group is denoted as “a”.</p