identification of the structural determinants of color variation. In this paper, we addressed two cases of color change. Two mutations turned out to be responsible for the unusual blue color in chromoprotein amilCP: S64C and S183T (Fig. 4). Residue 64 is immediately adjacent to the chromophore-forming triad, while the 183th side chain is involved in the interface between monomers within a tetrameric FP structure. Interestingly, position 64 is also occupied by cysteine in an artificially generated far-red emitting mutant of DsRed, mPlum [41]. There is unexpected epistatic interaction of these two mutations with the third one, I162L, which dramatically slows down the maturation of the chromo- protein if introduced alone, but does not have such an effect in combination with either S64C or S183T. Interestingly, the mutation I162L makes the protein slightly bluer if combined with S64C. From this it is reasonable to speculate that if the blue color was indeed the target of selection, the natural order of mutations most likely was S64C, I162L, S183T, resulting in a gradual transition towards the blue color. Two blue chromoproteins from sea anemones (order Acti- niaria): aeCP (absorption maximum 597 nm) [42] and the remarkable cjBlue (absorption maximum 610 nm) [43] must be mentioned here. All the Actiniaria chromoproteins belong to the Actiniaria-specific clade A, and thus clearly arose independently from coral chromoproteins. Similar to amilCP, both aeCP and cjBlue contain C64 and T183-but so do many other Actiniaria chromoproteins that are purple. It can be speculated that, although the structural determinants of blue color in aeCP and cjBlue may include the same residues that we identified in amilCP, the non-fluorescent color variation in Actiniaria is due to some other mutations that also contribute to the blue color. The second key spectrum-modifying mutation that we deter- mined is the glutamic acid in position 167, conferring a novel excitation property to cyan proteins presumably indicative of a neutral chromophore ground state (Fig. 5). Such a modification was previously unknown in cyan FPs, either wild-type or artificially generated mutant variants, although the residue at position 167 has been previously implicated in contributing to the cyan phenotype in general [12,44]. Neutral-chromophore cyan pro- teins, similar to GFP, may become valuable photoactivated markers [45] due to the proton transfer process characteristic of their photocycle [37]. Understanding the function of coral FPs Despite the great interest in discovering new FPs and adopting them for biotechnology needs, the progress in understanding their biological function (or functions) in non-bioluminescent organisms such as corals has been frustratingly slow. Currently there are several hypotheses based on indirect evidence, of which several or none may eventually turn out to be true. The ideas related to symbiosis with dinoflagellate algae of the genus Symbiodinium (zooxanthellae) include photoprotection (suggested by Kawaguti [46,47] and substantiated by physiology data by Salih and co- authors [48]), fine symbiosis regulation [12], aposematic colora- tion, and masking the presence of algal pigments within coral tissues from herbivorous fishes [8]. Alternative explanations include deactivation of reactive oxygen species [49] and proton pumping [50,51]. It should be noted that both of these latter hypotheses have been suggested based on the experiments with the original jellyfish-derived GFP, which has a neutral ground state chromophore and shows a peculiar proton transfer during the photocycle [37]. Until now neutral chromophores were not observed in coral FPs; however, this study reveals multiple such cases in cyan proteins. It is possible therefore that the proton- transfer photocycle, perhaps associated with either proton Coral Fluorescent Proteins pumping or reactive oxygen species deactivation, constitutes part of the function of the cyan color in particular. Our recent statistical phylogenetic analysis of FPs from Faviina, coupled with the site- directed mutagenesis study, revealed that the new non-green colors (cyan and red) evolved under the pressure of positive natural selection, which means that the diverse colors must serve some essential function [12]. Multiple events of parallel evolution of the same colors highlighted by this present work strongly corroborate this result. We also found previously that a subset of residues arranged as an intra-molecular interface in Faviina FPs evolved under diversifying positive selection, suggestive of a ‘‘co-evolu- tionary arms race’’ with an unknown binding partner [12]. Although we chose to interpret these observations in light of the symbiosis-related functionality, other explanations may be equally probable, involving functions unrelated to symbiosis, and perhaps even not related to fluorescence or any light modification (such as deactivation of oxygen radicals) if different colors translate into different reactive properties. To finally settle the question of the function of coral fluorescence a series of studies is necessary, dedicated specifically to finding the ecological correlates of coral fluorescence variation. Spatial and temporal patterns of protein and gene expression have to be analyzed, as well as the tissue distribution of individual color types. Preferably, such a study should be conducted across color morphs of a single coral species for which the full complement of FP colors has been cloned. The present work suggests a promising model for such kind of research: Acropora millepora, which yielded all four principal colors (cyan, green, red and non-fluorescent blue) and is an emerging genomic model [52,53]. Studies of genomic loci of coral GFP-like proteins may shed additional light on their evolutionary history, by generally improving the resolution of the phylogenetic tree and highlighting major transition events related to gene duplication and subfunctionalization. Such information will be invaluable for reconstructing the ancestral sequences and backtracking the phenotypic shifts, to get to the basics of color determination at the sequence level. Finally, very important for understanding the biological function of the coral GFPs will be to investigate their protein-protein interactions in vivo, which is especially interesting in relation to the putative molecular interface that is under positive natural selection [12]. Materials and Methods Collection of samples Samples (100–500 mg of tissue) of the Caribbean coral species (Agaricia fragilis, Scolymia cubensis, Eusmilia fastigiata, Montastrea cavernosa, and Porites porites) were obtained from the Florida Keys Marine Sanctuary under National Marine Sanctuary authorization FKNMS-2000-009. Samples of Indo-Pacific corals (Acropora aculeus, Acropora millepora, Acropora nobilis, Acropora eurostoma, Echinophyllia echinata, Echinopora forskaliana, Galaxea fascicularis, Goniopora djiboutiensis, Montipora efflorescens, Montipora millepora, Mycedium elephantotus, Psammo- cora sp., Stylocoeniella sp., and Stylophora pistillata) as well as soft coral Sarcophyton sp were collected at several locations on the Great Barrier Reef, Australia under Marine Parks Permit #G05/13283.1. Cloning and expression of coral FPs Total RNA was isolated from the organism using RNAqueous kit (Ambion) and amplified cDNA was prepared from it using SMART protocol [54]. The complete cDNA coding sequences for GFP-like proteins were obtain by modified Step-Out RACE [55,56] using degenerate primers for homology cloning. For 5’ stage of RACE, 12 pairs of upstream-directed degenerate primers were used: PLoS ONE | www.plosone.org 10 July 2008 | Volume 3 | Issue 7 | e2680