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...

Vertebrate pigmentation is a highly diverse trait mainly determined by neural crest cell derivatives. It has been suggested that two rounds (1R/2R) of whole-genome duplications (WGDs) at the basis of vertebrates allowed changes in gene regulation associated with neural crest evolution. Subsequently, the teleost fish lineage experienced other WGDs, including the teleost-specific Ts3R before teleost radiation and the more recent Ss4R at the basis of salmonids. As the teleost lineage harbors the highest number of pigment cell types and pigmentation diversity in vertebrates, WGDs might have contributed to the evolution and diversification of the pigmentation gene repertoire in teleosts. We have compared the impact of the basal vertebrate 1R/2R duplications with that of the teleost-specific Ts3R and salmonid-specific Ss4R WGDs on 181 gene families containing genes involved in pigmentation. We show that pigmentation genes (PGs) have been globally more frequently retained as duplicates than other genes after Ts3R and Ss4R but not after the early 1R/2R. This is also true for non-pigmentary paralogs of PGs, suggesting that the function in pigmentation is not the sole key driver of gene retention after WGDs. On the long-term, specific categories of PGs have been repeatedly preferentially retained after ancient 1R/2R and Ts3R WGDs, possibly linked to the molecular nature of their proteins (e.g., DNA binding transcriptional regulators) and their central position in protein-protein interaction networks. Taken together, our results support a major role of WGDs in the diversification of the pigmentation gene repertoire in the teleost lineage, with a possible link with the diversity of pigment cell lineages observed in these animals compared to other vertebrates.

Thyroid hormones modulate not only multiple functions in vertebrates (energy metabolism, central nervous system function, seasonal changes in physiology, and behavior) but also in some non-vertebrates where they control critical post-embryonic developmental transitions such as metamorphosis. Despite their obvious biological importance, the thyroid hormone precursor protein, thyroglobulin (Tg), has been experimentally investigated only in mammals. This may bias our view of how thyroid hormones are produced in other organisms. In this study we searched genomic databases and found Tg orthologs in all vertebrates including the sea lamprey (Petromyzon marinus). We cloned a full-size Tg coding sequence from western clawed frog (Xenopus tropicalis) and zebrafish (Danio rerio). Comparisons between the representative mammal, amphibian, teleost fish, and basal vertebrate indicate that all of the different domains of Tg, as well as Tg regional structure, are conserved throughout the vertebrates. Indeed, in Xenopus, zebrafish, and lamprey Tgs, key residues, including the hormonogenic tyrosines and the disulfide bond-forming cysteines critical for Tg function, are well conserved despite overall divergence of amino acid sequences. We uncovered upstream sequences that include start codons of zebrafish and Xenopus Tgs and experimentally proved that these are full-length secreted proteins, which are specifically recognized by antibodies against rat Tg. By contrast, we have not been able to find any orthologs of Tg among non-vertebrate species. Thus, Tg appears to be a novel protein elaborated as a single event at the base of vertebrates and virtually unchanged thereafter.

Ecological disturbance and climate are key drivers of temporal dynamics in the demography and genetic diversity of natural populations. Microscale refuges are known to buffer species' persistence against environmental change, but the effects of such refuges on demographic and genetic patterns in response to short-term environmental variation are poorly understood. We quantified demographic and genetic responses of mountain brushtail possums (Trichosurus cunninghami) to rainfall variability (1992-2013) and to a major wildfire. We hypothesized that there would be underlying differences in demographic and genetic processes between an unburnt mesic refuge and a topographically exposed zone that was burnt in 2009. Fire caused a 2-year decrease in survival in the burnt zone, but the population grew after the fire due to immigration, leading to increased expected heterozygosity. We documented a fire-related behavioural shift, where the rate of movement by individuals in the unburnt refuge to the burnt zone decreased after fire. Irrespective of the fire, there were long-term differences in demographic and genetic parameters between the mesic/unburnt refuge and the nonmesic/burnt zone. Survival was high and unaffected by rainfall in the refuge, but lower and rainfall-dependent in the nonmesic zone. Net movement of individuals was directional, from the mesic refuge to the nonmesic zone, suggesting fine-scale source-sink dynamics. There were higher expected heterozygosity (HE ) and temporal genetic stability in the refuge, but lower HE and marked temporal genetic structure in the exposed habitat, consistent with reduced generational overlap caused by elevated mortality and immigration. Thus, fine-scale refuges can mediate the short-term demographic and genetic effects of climate and ecological disturbance.

The emergence of the steroid system is coupled to the evolution of multicellular animals. In vertebrates in particular, the steroid receptor repertoire has been shaped by genome duplications characteristic to this lineage. Here, we investigate for the first time the composition of the androgen receptor–signaling pathway in ray-finned fish genomes by focusing in particular on duplicates that emerged from the teleost-specific whole-genome duplication. We trace lineage- and species-specific duplications and gene losses for the genomic and nongenomic pathway of androgen signaling and subsequently investigate the sequence evolution of these genes. In one particular fish lineage, the cichlids, we find evidence for differing selection pressures acting on teleost-specific whole-genome duplication paralogs at a derived evolutionary stage. We then look into the expression of these duplicated genes in four cichlid species from Lake Tanganyika indicating, once more, rapid changes in expression patterns in closely related fish species. We focus on a particular case, the cichlid specific duplication of the rac1 GTPase, which shows possible signs of a neofunctionalization event.

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