Diversity and Evolution of Coral Fluorescent Proteins

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.

GFP样荧光蛋白（GFP-like fluorescent proteins，FPs）是造礁珊瑚（珊瑚纲（Anthozoa）、石珊瑚目（Scleractinia））的关键颜色决定因子，且作为潜在的基因编码荧光标记物受到广泛关注。本研究报道了来自珊瑚的40个新增GFP家族成员。珊瑚FPs存在三大主要旁系同源谱系：其中一个谱系保留于所有采样珊瑚类群中，负责非荧光紫蓝色表型；另外两个谱系则分别演化出完整的典型珊瑚荧光色彩组合（青色、绿色与红色），并在珊瑚类群间发生了分化分选。在新克隆的蛋白中，来自蜂巢珊瑚科（Faviidae）的棘星珊瑚（Echinopora forskaliana）的"发色红"色型，以及来自杯形珊瑚科（Pocilloporidae）的柱形珊瑚（Stylophora pistillata）的粉色发色蛋白，均独立于其他珊瑚发色蛋白演化而来。有数种青色FPs具有新型激发光谱，表明其发色团处于中性基态，这一特性由残基E167决定（编号参照维多利亚水母（A. victoria）来源的GFP）。来自微孔珊瑚（Acropora millepora）的发色蛋白呈现出罕见的蓝色而非紫色，这一表型由两个突变导致：S64C与S183T。本研究采用新型概率采样方法，重构了所有珊瑚FPs的共同祖先，以及蜂巢珊瑚亚目（Faviina）三大主要荧光色彩类群的更衍生共同祖先。这两个重构蛋白均呈绿色，这与珊瑚纲以外类群中发现的荧光蛋白颜色一致。有趣的是，该全珊瑚祖先蛋白中有相当一部分的发色团似乎被锁定在非荧光中性状态，这或许反映了珊瑚FPs演化早期实现快速色彩多样化的过渡阶段。本研究结果阐明了珊瑚色彩多样性的趋同与平行演化程度，为揭示驱动色彩多样化的演化过程提供了实验研究基础，同时也为不同色彩的结构决定因子的比较分析提供了可行路径。