Diversity and Evolution of Coral Fluorescent Proteins Naila O. Alieva1, Karen A. Konzen2, Steven F. Field1, Ella A. Meleshkevitch2, Marguerite E. Hunt1, Victor Beltran-Ramirez3, David J. Miller3, Jo¨ rg Wiedenmann4,5, Anya Salih6, Mikhail V. Matz1* 1 Section of Integrative Biology, University of Texas at Austin, Austin, Texas, United States of America, 2 Whitney Laboratory for Marine Bioscience, University of Florida, Saint Augustine, Florida, United States of America, 3 ARC Centre of Excellence in Coral Reef Studies, James Cook University, Townsville, Queensland, Australia, 4 National Oceanography Centre, University of Southampton, Southampton, United Kingdom, 5 Institute of General Zoology and Endocrinology, University of Ulm, Ulm, Germany, 6 School of Natural Sciences, University of Western Sydney, Penrith South DC, New South Wales, Australia Abstract GFP-like fluorescent proteins (FPs) are the key color determinants in reef-building corals (class Anthozoa, order Scleractinia) and are of considerable interest as potential genetically encoded fluorescent labels. Here we report 40 additional members of the GFP family from corals. There are three major paralogous lineages of coral FPs. One of them is retained in all sampled coral families and is responsible for the non-fluorescent purple-blue color, while each of the other two evolved a full complement of typical coral fluorescent colors (cyan, green, and red) and underwent sorting between coral groups. Among the newly cloned proteins are a ‘‘chromo-red’’ color type from Echinopora forskaliana (family Faviidae) and pink chromoprotein from Stylophora pistillata (Pocilloporidae), both evolving independently from the rest of coral chromoproteins. There are several cyan FPs that possess a novel kind of excitation spectrum indicating a neutral chromophore ground state, for which the residue E167 is responsible (numeration according to GFP from A. victoria). The chromoprotein from Acropora millepora is an unusual blue instead of purple, which is due to two mutations: S64C and S183T. We applied a novel probabilistic sampling approach to recreate the common ancestor of all coral FPs as well as the more derived common ancestor of three main fluorescent colors of the Faviina suborder. Both proteins were green such as found elsewhere outside class Anthozoa. Interestingly, a substantial fraction of the all-coral ancestral protein had a chromohore apparently locked in a non-fluorescent neutral state, which may reflect the transitional stage that enabled rapid color diversification early in the history of coral FPs. Our results highlight the extent of convergent or parallel evolution of the color diversity in corals, provide the foundation for experimental studies of evolutionary processes that led to color diversification, and enable a comparative analysis of structural determinants of different colors. Citation: Alieva NO, Konzen KA, Field SF, Meleshkevitch EA, Hunt ME, et al. (2008) Diversity and Evolution of Coral Fluorescent Proteins. PLoS ONE 3(7): e2680. doi:10.1371/journal.pone.0002680 Editor: Hany A. El-Shemy, Cairo University, Egypt Received March 25, 2008; Accepted June 15, 2008; Published July 16, 2008 Copyright: 2008 Alieva et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the NIH grant R01 GM66243 to M.V.M., the Australian Research Council (ARC) via the Centre of Excellence for Coral Reef Studies to D.J.M, Deutsche Forschungsgemeinschaft grant Wi1990 2-1 to J.W., and ARC/NHMRC Network FABLS Australia (collaborative grant to A.S. et al.) Competing Interests: The authors have declared that no competing interests exist. * E-mail: matz@mail.utexas.edu Introduction Fluorescent proteins (FPs) homologous to the green fluorescent protein (GFP) from the jellyfish Aequorea victoria are a fascinating protein family in many respects. Being only about 230 amino acid residues long, coral FPs, during their evolution, acquired an ability to synthesize several distinct types of fluorescent or colored moiety–the chromophore–from their own residues in two or three consecutive autocatalytic reactions, resulting in sometimes dra- matically different spectroscopic characteristics [1]. Since the first description of Anthozoan members of the GFP family, these proteins have given rise to a variety of in vivo imaging techniques capitalizing on their unique spectral, physical or biochemical properties [2,3,4]. The ease with which coral FPs can be expressed and screened for phenotypic changes makes them ideal models for experimental studies in evolution of protein families, addressing in particular such important questions as convergent molecular evolution and the origins of molecular complexity [5,6]. Last but not least, coral FPs are major determinants of the coral reef color diversity [7,8,9,10], accounting for practically every visible coral color other than the brown of the photosynthetic pigments of algal symbionts (possible exception is the non-fluorescent yellow in some representatives of Poritidae and Dendrophylliidae that may be due to melanin-related pigments; C. Palmer, pers. comm.). A suggestion that the red appearance of some corals may be predominantly due to the phycoerythrins of cyanobacterial symbionts rather than intrinsic GFP-like proteins [11] was not supported in subsequent experiments [10]. FPs are the only known natural pigments in which the color is determined by the sequence of a single gene, which provides a unique opportunity to directly study the evolution of coral reef colorfulness at the molecular level [12]. Previous studies revealed four basic colors of coral FPs: three fluorescent ones (cyan, green, and red) and a non-fluorescent one (purple-blue) [9,13]. Of these, only green and cyan share the same chromophore structure [14]. There are two types of red chromophore representing alternative ways to extend the ‘‘green’’ structure by means of an additional autocatalytic reaction. These chromophore types can be called DsRed-type [15] and Kaede- type [16] after the first proteins in which they were found. DsRed- like and Kaede-like chromphores are easily discernable by the shape of the excitation and emission spectra: Kaede-type proteins show much narrower major peaks with smaller Stokes shifts and a characteristic shoulder at 630 nm in the emission spectrum that makes them look remarkably like cyanobacterial phycoerythrins PLoS ONE | www.plosone.org 1 July 2008 | Volume 3 | Issue 7 | e2680