Figure 4. Positions of emission maxima in the mutated purple chromoprotein gfasCP in comparison to the blue amilCP. Horizontal axis is wavelength in nanometers, the bars indicate the position of the absorption peak in the mutant. The colors of the bars approximately correspond to the colors of the mutants. Mutations S64C and S183T were found to be responsible for the blue color in amilCP (numeration according to GFP). Mutation I162L results in a very slowly maturing protein, hence pale color of the corresponding bar. This effect is rescued by any of the other two mutations. doi:10.1371/journal.pone.0002680.g004 induction of the expression in bacteria to develop color to the intensity comparable to what was seen already overnight in other variants. This effect was completely rescued by either of the two color-affecting mutations. Neutral versus anionic chromophore in cyan proteins Two novel cyan proteins from clade C, psamCFP and mmilCFP, feature an excitation spectrum very similar in shape to the wild-type GFP from Aequorea victoria, with the major peak at 404 nm (Fig. 1 and Table 1). It is very likely that such a spectrum, by analogy to GFP, indicates the predominantly neutral ground-state of the chromo- phore. In addition, in acroporid cyans (anobCFP and amilCFP) the excitation curve seems to contain a blue-shifted component, suggestive of a possible presence of the neutral chromophore in these proteins as well (Fig. 1 and 5). We noticed that in all these proteins, unlike all other FPs, the position 167 (GFP numeration) is Coral Fluorescent Proteins occupied by glutamic acid. In a closely related cyan protein meffCFP, which does not have the 404 nm excitation band, position 167 is occupied by glycine. We mutated the residue 167 to glutamic acid in meffCFP and to glycine in anobCFP. In the former case the shortwave excitation band appeared and in the latter it vanished (Fig. 5), thus confirming the role of E167 in conferring the shortwave-excitation phenotype that is most likely associated with the neutral chromophore ground state. Discussion FP phylogeny versus host organism phylogeny A substantial number of FPs from organisms not belonging to the order Scleractinia are intermingled within the three coral clades with high phylogenetic support. These include three other orders of hexacorals (sub-class Zoantharia), Corallimorpharia, Zoanthidea and Actiniaria–as well as, unexpectedly, order Alcyonacea (soft corals) from another sub-class (octocorals, Alcyonaria) (Fig. 2). Alcyonacea placement received additional support as a result of the present study in the form of yet one more protein , green sarcGFP, cloned from an Alcyonacea representative Sarcophyton sp. that groups together with the two previously known Alcyonacea FPs (clavCFP and dendRFP) within clade D. This is in strong contradiction with the current taxonomy that calls for the separation of subclasses (Alcyonaria and Zoantharia) preceding the separation of Zoantharia orders (Actiniaria, Zoanthidea, Corallimorpharia and Scleractinia). There are three ways to explain the FP/taxonomy incongruence: (i) spurious taxonomy; (ii) sorting of ancient paralogous gene lineages and (iii) horizontal gene transfer. Unresolved taxonomic relationships between Scleractinia and Corallimorpharia may account for most of the discordance involving these two orders. Scleractinia have been proposed to originate several times from a Corallimorpharia-like ancestor by acquiring the ability to deposit a calcium carbonate skeleton [38]. More recent molecular analysis suggested a different scenario where Corallimorpharia originate once within Scleractinia by means of losing the skeleton [39]. Placement of Corallimorpharia proteins among Scleractinia is therefore expected. The polyphy- letic origin of Scleractinia could also be responsible for the curious pattern of sorting of coral suborders between FP clades. On the basis of a combination of molecular and morphology characters at least two separate origins of Scleractinia have been proposed [31,40]. These two groups of corals do not correspond to the traditional classification by suborders and have been named Figure 5. Glutamic acid at position 167 (numeration according to GFP) determines the ‘‘neutral-chromophore’’ phenotype in novel cyan fluorescenr proteins. A: Replacement of native E 167 by glycine in anobCFP leads to disappearance of 425 nm excitation peak and shift of both excitation and emission curves towards green. B: Reciprocal replacement G167E in a cyan protein meffCFP leads to the opposite results: virtually all the protein bulk started absorbing at 400 nm. Notably, the emission peak did not change. doi:10.1371/journal.pone.0002680.g005 PLoS ONE | www.plosone.org 8 July 2008 | Volume 3 | Issue 7 | e2680