Actinopterygian fishes harbor at least eight distinct pigment cell types, leading to a fascinating diversity of colors. Among this diversity, the cellular origin of the white color appears to be linked to several pigment cell types such as iridophores or leucophores. We used the clownfish Amphiprion ocellaris, which has a color pattern consisting of white bars over a darker body, to characterize the pigment cells that underlie the white hue. We observe by electron microscopy that cells in white bars are similar to iridophores. In addition, the transcriptomic signature of clownfish white bars exhibits similarities with that of zebrafish iridophores. We further show by pharmacological treatments that these cells are necessary for the white color. Among the top differentially expressed genes in white skin, we identified several genes (fhl2a, fhl2b, saiyan, gpnmb, and apoD1a) and show that three of them are expressed in iridophores. Finally, we show by CRISPR/Cas9 mutagenesis that these genes are critical for iridophore development in zebrafish. Our analyses provide clues to the genomic underpinning of color diversity and allow identification of new iridophore genes in fish.

Color patterns provide easy access to phenotypic diversity and allow the questioning of the adaptive value of traits or the constraints acting on phenotypic evolution. Reef fish offer a unique opportunity to address such questions because they are ecologically and phylogenetically diverse and have the largest variety of pigment cell types known in vertebrates. In addition to recent development of their genetic resources, reef fish also constitute experimental models that allow the discrimination of ecological, developmental, and evolutionary processes at work. Here, we emphasize how the study of color patterns in reef fish can be integrated in an Eco/Evo/Devo (ecological evolutionary developmental) perspective and we illustrate that such an approach can bring new insights on the evolution of complex phenotypes. Why Study Reef Fish and Their Color Patterns?Questions regarding the diversity, evolution, and ecological significance of color patterns (see Glossary) have caught scientists' attention for centuries [1]. Pigmentation has been studied using a wide variety of animal models from hexapods to vertebrates [1,2]. Fruit flies and mice are still important models to study pigmentation genes [3] but, over the last few years, teleost fish have also became efficient systems for addressing questions related to color patterns. Zebrafish and medaka are helpful models for combining genetic manipulations with live imaging, and their study has provided new insights on the cellular and molecular mechanisms that drive the development of color patterns [4]. Other fish such as cichlids and guppies have also provided valuable insight into genes and molecular mechanisms underlying specific traits (egg spots and stripes) and various color ornaments [5][6][7].While mammals only possess melanocytes, the teleost lineage harbors the highest number of pigment cell typesalso called chromatophores (e.g., melanophores, xanthophores, and iridophores) [8]. This diversity can explain the diversity of color and their patterns and implies the involvement of many pigmentation genes. The list of identified genes has increased in recent years (Box 1) and the whole genome duplication that occurred at the basis of the teleost lineage has been identified as a major contributor to this diversity [9].To be able to fully understand the evolution of traits such as those displayed in color patterns and the genetic mechanisms underlying the responses of organisms to their natural environment, it is important to perform Eco/Evo/Devo approaches. However, ecological and behavioral roles of color patterns have not been studied in the model organisms cited above, leading to a black box concerning how proximate factors shape color patterns and their diversity over evolution. Reef fish offer promising models to address such questions because they do express much of the amazing diversity of color patterns as well as associated behavioral and ecological variation. Their original color patterns include dark or conspicuous colors, and can be made of a diverse combin...