Phylogenetic analysis and the independent origins of PSBS and LHC families. (A) Four-cluster likelihood mapping analysis  of individual CB motifs in LHC and PSBS proteins showing the likelihood support for three alternative topologies. 64 amino acid positions (32 per CB motif) from 60 protein sequences including all known LHC subfamilies  and the complete PSBS diversity were analyzed. The topology with LHC helix I as sister group to LHC helix III is strongly supported with 94.1% over the one expected under a common origin with 2.5% in the three-partite diagram (or 84.2% over 0.2% in the seven-partite diagram). (B) Schematic diagram of a phylogenetic analysis based on the conserved sequence motifs of the first and third TM helices of PSBS and LHC proteins. A selection of 22 diverse SEP sequences from glaucophytes, red algae and the green lineage was included. The alignment contained 31 unambiguously aligned amino acid positions. The distinct clustering of the different LHC and PSBS helices provides corroborating evidence that LHC sequences do not share a common four-helix ancestor with PSBS sequences. The shown topology was significantly (p = 0.0001) supported over the alternative topology (Additional file 1, Figure S1B), analyses were done in TreePuzzle. The true tree is presented in Additional file 1, Figure S2. (C) Hypothesis for the independent origin of LHC and PSBS proteins from distinct SEP ancestors according to the present study. LHC proteins likely evolved from a SEP with a putative carotenoid-binding motif (orange box), which was duplicated together with the CB motif in an internal gene duplication/unequal crossing-over of tandem genes. PSBS protein evolved from a different ancestor from an ancient pool of paralogous SEP members. PSBS protein has highly conserved second and fourth helices (dark grey boxes). Note that following this hypothesis, helices I and III of the resulting LHC and PSBS proteins must be most similar within the same proteins, which is in agreement with (A) and (B) but in conflict with a previously suggested scenario (Additional file 1, Figure S1).