[19,30] rather than a wild-type DsRed-like fluorescent protein, and given its isolated phylogenetic position (see below), we believe that it is warranted to recognize this protein as a representative of its own new color class, ‘‘chromo-red fluorescent protein’’. Phylogeny The phylogenetic tree for Cnidarian fluorescent proteins is presented on Fig. 2. Scleractinian FPs form three separate clades, which we earlier designated B through D [13]. Each of these clades has a strong phylogenetic support (posterior probability essentially equals one), although the relationship between them remains unresolved. The grouping of these three clades to the exclusion of all but one FP from Actiniaria (clade A), Pennatu- lacea, and Ceriantharia is also very highly supported. Of the three Scleractinia clades, clade B is a clear example of a separate paralogous lineage that is retained in the genome, despite presence of multiple other FP genes, due to functional specialization. This clade contains non-fluorescent purple-blue chromoproteins from nearly every sampled family of Scleractinia (plus some from Corallimorpharia). Clades C and D underwent sorting among coral groups, so that we never find representatives of both within a single coral species. Each of these two clades contains a full complement of typical coral fluorescent colors: green, cyan, and red. Notably, the red fluorescent proteins of clade C are all DsRed-type, whereas they are Kaede-type in clade D. The specrtroscopically unique chromo-red protein eforCP/RFP from Echinopora forskaliana does not belong to any of the three major clades, although it constitutes their sister group. Some of the relationships between coral FPs resemble patterns suggested by the novel molecular-based coral phylogeny [31,32]; however, its most basic subdivision into Robusta and Complexa is not recapitulated, most likely due to the extensive lineage sorting in the FP family. Clade B. This clade is comprised mostly of the purple-blue non-fluorescent chromoproteins, which have been cloned from families Acroporidae, Pocilloporidae, Poritidae, Faviidae, Pectinidae, Oculinidae, and Dendrophyliidae. Most of these families yielded other FPs situated elsewhere within the tree. The tree on Fig. 2 includes only a small subset of the known chromoproteins, which we first describe in this paper. Omitting the others does not affect the overall phylogeny since all the chromoproteins of clade B are unusually similar in sequence, even the ones from different orders, Scleractinia and Corallimorpharia. Multiple, very similar chromoproteins can often be identified within a single species [33], suggesting a possibility of concerted evolution that may contribute to their sequence conservation. In addition to chromoproteins, clade B contains a group of corallimorpharian FPs, two of which are DsRed-type reds (including DsRed itself) and one cyan, plus a novel red FP from Porites porites (pporRFP) that occupies the most basal position within the clade and is also of the DsRed type. Thus far, clade B does not include any green FPs, which suggests that the common ancestor of this clade might have been either a red FP or a chromoprotein. Whether this is true or not, the grouping of all but one coral chromoproteins within one clade unequivocally indicates that the paralogous gene lineage responsible for the purple-blue color originated before the separation of scleractinian families. Clade C. This clade received significant expansion through addition of the proteins reported here, as well as cloned by other laboratories since 2002. Ironically, clade C originally contained only the proteins from order Zoanthidea and the cyan protein from Anemonia majano (order Actiniaria), the placement of which within this clade we now tend to view as a phylogenetic complication (see Discussion). All of the other 24 proteins that joined clade C as a result of recent studies came from the order Coral Fluorescent Proteins Scleractinia. Clade C includes three well-supported subclades (C1, C2 and C3, Fig. 2) each of which contains its own events of color diversification. C1 subclade unites representatives from coral families Fungiidae (suborder Fungiina), Meandrinidae (Meandriina) and Rhizangiidae (Faviina), which may correspond to a grouping of these families into one of the Robusta subclades in the novel coral phylogeny [32]. C1 features diversification into cyan, green, and DsRed-type red fluorescent colors. At the divergence point of subclades C2 and C3 there is a surprise: the pink chromoprotein spisCP from Stylophora pistillata (suborder Archaeocoeniina, family Pocilloporidae). This protein clearly has evolved independently from the rest of coral chromoproteins. Interestingly, other representatives of the same coral family (but not of the same genus) yielded ‘‘conventional’’ chromoproteins of the clade B affiliation. Subclade C2 contains green and cyan proteins from Archae- ocoeniina suborder (families Acroporidae and Pocilloporidae) plus a cyan protein from sea anemone Anemonia majano (amajCFP, original name amFP486). Notable in this subclade are the multiple splits between cyan and green lineages: apparently these colors evolved from each other several times. The C3 subclade is again a mixture of coral suborders: it contains a green protein from Porites porites (suborder Poritiina), cyan from Psammocora sp. (Fungiina) and one green and two red proteins from Acroporidae family (Archaeocoeniina); plus a group of proteins from order Zoanthidea. C3 is the most controversial subclade in the whole tree: its composition cannot be reconciled with any of currently considered phylogenies (see Discussion below). A notable feature of the subclade C3 is the secondary color radiation within Zoanthidea branch. The three Zoanthidea sequences correspond to red, yellow and green protein of which the red is basal; moreover, red fluorescent proteins amilRFP and meffRFP occupy the sequential basal positions with respect to the Zoanthidea branch. This renders it most likely that common ancestor of all the Zoanthidea proteins was a fluorescent red protein. Clade D. Clade D includes several well-resolved nested subclades. The most basal branch corresponds to the green protein from coral genus Agaricia (suborder Fungiina). Moving up clade D, there is a group of FPs from order Corallimorpharia (mushroom anemones) and, rather unexpectedly, a group from order Alcyonaria (soft corals). The rest of clade D contains only FPs from families Faviidae, Mussidae, Trachyphyllidae, Oculinidae, and Pectinidae, all belonging to the suborder Faviina. With the exception of FPs from genus Galaxea (family Oculinidae), these proteins fall into three groups corresponding to cyan, green, and red fluorescent colors, of which cyan and red are monophyletic and green–paraphyletic. Gene conversion Within the C3 subclade, there is an obvious case of gene conversion between green and red proteins of Montipora efflorescens (meffGFP and meffRFP): these two proteins are identical starting with the residue 66 (according to GFP numeration; in fact it is the chromophore-forming tyrosine) with not even a single third codon position substitution, whereas the N-terminal parts are substan- tially different (76% identity over 198 nucleotides of the corresponding coding region). The existence of such transcripts in the original Montipora efflorescens RNA sample was confirmed through independent RT-PCRs with gene-specific primers followed by sequencing of the product. Comparison to the closely related red fluorescent protein from Acropora millepora (amilRFP) revealed that amilRFP coding region is 90% identical to the 498 nucleotides of the converted meffRFP/meffGFP portion. Exactly the same level of identity is found between amilRFP and meffRFP PLoS ONE | www.plosone.org 5 July 2008 | Volume 3 | Issue 7 | e2680