Complexa and Robusta referring to the prevailing mode of skeleton deposition [32]. There is some resemblance of this novel phylogeny in the FP tree, such as the C1 subclade uniting FPs from Fungiidae, Rhizangiidae and Meandrinidae (Robusta), close positioning (although not as sister groups) of Agariciidae and Oculinidae (Complexa) within clade D, as well as grouping of Poritiidae and Acroporidae (Complexa) within subclade C3. However, the FP phylogeny does not generally recapitulate the Complexa-Robusta split: FPs from groups that are thought to belong to Complexa (Poritiidae, Acroporidae, Agariciidae and Oculinidae) show no tendency to cluster into a unique clade (Fig. 2). For example, FPs from ‘‘robust’’ Pocilloporidae family (pdamCFP and spisCP) fall within subclade C2/C3 alongside the sequences from ‘‘complex’’ Acroporidae and Astrocoeniidae, seemingly in accord with the traditional taxonomic grouping of these families within Archaeocoeniina suborder. We think that the best explanation for most of these discrepancies is paralogous lineage sorting. This explanation assumes that gene divergence within the ancestral genome preceded the organismal divergence. For example, to account for the occurrence of Alcyonaria proteins within clade D as well as deeper within the phylogeny (FPs from order Pennatulacea, Fig. 2) without the need to invoke pervasive polyphyly of Anthozoa orders, one may assume that the diversity of sequences bracketed by these two occurrences (i.e., all the major Zoantharia clades, from A to D) existed as paralogous genes within the genome of the common ancestor of Zoantharia and Alcyonaria [5]. The multiplicity of closely related genes accounting for each basic color in a closely investigated great star coral Montastrea cavernosa [9] suggests that the rate of gene duplication in the coral GFP-like gene family is indeed very high. The FP phylogeny may be predominantly reflecting the process of gene birth and death interspersed by selective sweeps leading to novel spectral features [12], which may considerably obscure the phylogenetic signal form the host organism evolution. A group of sequences that does not quite fit any of the above explanations are Zoanthidea FPs, occupying a surprising position among scleractinian FPs within subclade C3 (Fig. 2). Unlike Corallimorpharia, order Zoanthidea was never suggested to have originated within Scleractinia by any analysis, so taxonomic uncertainty is not likely to be the case here. On the other hand, the position of Zoanthidea FPs within the FP tree is probably too derived to plausibly evoke the paralogous sorting explanation. In this case, it would require assuming a very unlikely scenario in which most of the FP diversity evolved as paralogous lineages in the common ancestor of Anthozoa orders and not much evolution happening since then. Zoanthidea FPs are not an artifact resulting from contamination by Scleractinian material, since the first Zoanthidea proteins were isolated before any coral material was searched for FPs, at least in our lab [23]. It is tempting to speculate that Zoanthidea acquired the FP gene from Scleractinia relatively recently via horizontal gene transfer, which may have been mediated by a common symbiont or pathogen. It is possible that some evidence of this event may be obtained through comparison of the genomic context of FP genes in Zoanthids and corals. Ancestral colors Understanding the order and direction of the color transitions within the FP phylogeny is very important for studies of the structural determinants of color. To identify these, a typical comparative approach considers amino acid differences between the two most closely related proteins of different colors. However, in addition to the sites that are responsible for the color difference such a comparison will also reveal changes that were either neutral or related to a modification of other properties rather than color in Coral Fluorescent Proteins both lineages since their separation. To narrow down the search, it is possible to compare the present-day proteins not to each other, but to their common ancestor. This at once removes half (on average) of the ‘‘ballast’’ mutations from consideration since only one of the two evolutionary lines of descent is considered. There is also an additional benefit of having the reconstructed ancestral proteins available for site-directed mutagenesis studies. Mutagen- esis of present-day proteins can verify whether identities of certain residues are essential for the color; however, only changing these residues in the reconstructed ancestral protein in the evolutionary- forward direction can prove that such modifications are also sufficient [12]. We therefore reconstructed two ancestral proteins, all-coral ancestor and all-Faviina ancestor, which provide perspective to the history of coral color evolution. We found that both ancestral proteins, the one at the root of the whole coral FP diversity as well as the much more derived protein ancestral to all Faviina FPs, were green and virtually identical in their excitation-emission properties (Fig. 3 D and E), although the all-coral ancestor had a peculiar absorbance spectrum indicative of the presence of the chromophore in a dark neutral state (Fig. 3 C). Such remarkable stability of ancestral fluorescence phenotype over considerable evolutionary distance is rather surprising, considering that in the present dataset a substantial number of non-green proteins appear very shortly after the diversification of the three major coral FP clades (B–D). These include the whole of clade B that does not have any green members, the chromo-red protein eforCP/ RFP, the unusual pink chromoprotein spisCP that branches off early within clade C, as well as the red protein from Corallimorpharia that appears in the subclade that splits off in between the two reconstructed ancestral nodes (Fig. 2). It is reasonable to expect therefore that most of the coral FP tree has a ‘‘green trunk’’, i.e., that nearly every ancestral protein that had green descendants was green. One likely exception from this rule may be Zoanthidea proteins, which conceivably evolved from a red fluorescent protein since they arise from within a group of red FPs within C3 subclade (Fig. 2). The evolution of green from red is achievable simply by inhibition of the third stage of autocatalysis during the red chromophore synthesis [15]. The appearance of the unique three-ring yellow chromophore in zoanYFP [22] also becomes less surprising if it is viewed as a result of deviation from the already complex pathway of the red chromophore formation. Given the diversity of chromophores in Zoanthidea FPs despite high sequence similarity, addressing this particular case of color diversification will be a promising subject for a future in-depth study. The evolutionary significance of the strange absorption spectrum of the all-coral ancestor (Fig. 3C) is unclear at the moment, since none of its descendants show anything similar. It is tempting to speculate that this unusual phenotype reflects an important transitional stage that enabled quick diversification into a variety of colors early in the history of coral FPs. However, it is still possible that such an ancestral phenotype is, after all, a result of some unidentified systematic bias in the ancestral sequence prediction algorithm. Further ancestral reconstruction studies as well as in- depth structure-function analysis of the all-coral ancestral protein (beyond the scope of this paper) will clarify this issue. It is important to add that the phenotype of the all-Faviina ancestral protein reported here was identical to the previously reconstructed version of the same node based on much less sequence data [6]. This indicates that our ancestral reconstruction results are robust to the inclusion of new sequences into the phylogeny. Structural determinants of color variation The current dataset provides rich material for reconstruction of the evolutionary paths resulting in novel spectral features and PLoS ONE | www.plosone.org 9 July 2008 | Volume 3 | Issue 7 | e2680