Fig. 4. The leader sequence is defined by plastid ultrastructure rather than by its phylogeny. (A) Alignment of signal peptides from (top to bottom) five dinoflagellate (Amphidinium carterae, Symbiodinium sp and Gonyaulax polyedra) and six Euglena gracilis plastid-directed, nuclear-encoded proteins. Two conserved hydrophobic regions (blue) are interspersed with polar (yellow), acidic (red) or basic (green) residues. Gaps (white) were introduced to align the hydrophobic regions. The AXA signal peptidase site is at the end of the first hydrophobic region (arrow). (B) A phylogenetic reconstruction using glyceraldehyde-3-phosphate dehydrogenase sequences shows significant bootstrap support for the apicomplexan Toxoplasma and the dinoflagellate Gonyaulax as sister clades, using both plastid and cytoplasmic isoforms, as well as strong bootstrap support excluding Euglena. Numbers at the nodes indicate bootstrap support (10,000 trees). (C) Plastid evolution. Dinoflagellates and apicomplexans share a common ancestor both for the host cell and the plastid, and are unrelated to either Euglenoids or their plastids. Gene transfer from the endosymbiont nucleus to the new host cell nucleus must be accompanied by addition of a peptide signal, which allows the plastid-directed proteins to pass the new membranes. (D) Representative hydrophobicity plots of signal peptide sequences from the apicomplexan ribosomal protein S9 (RPS9), showing only a single hydrophobic region, as well as from a dinoflagellate and a Euglenoid glyceraldehyde-3-phosphate dehydrogenase (GAP), showing two distinct hydrophobic regions. All plots are to the same scale, with hydrophobicity increasing above the midline.