Anemonefish are better taxonomists than humans.

The symbiosis between giant sea anemones, algae of the family Symbiodiniaceae, and anemonefish is an iconic example of a mutualistic trio1,2. Molecular analyses have shown that giant sea anemones hosting anemonefish belong to three clades: Entacmaea, Stichodactyla, and Heteractis3,4,5 (Figure 1A). Associations among 28 species of anemonefish and 10 species of giant sea anemone hosts are complex. Some fish species are highly specialized to only one anemone species (e.g., Amphiprion frenatus with Entacmaea quadricolor), whereas others are more generalist (e.g., Amphiprion clarkii)1,2,6. Reasons for host preferences are obscured, among other things, by the lack of resolution in the giant sea anemone phylogeny. Here, we generated a transcriptomic dataset from 55 sea anemones collected from southern Japan to reconstruct these phylogenetic relationships. We observed that the bubble-tip sea anemone E. quadricolor, currently considered a single species, can be separated into at least four cryptic lineages (A-D). Surprisingly, these lineages can be precisely distinguished by observing their association with anemonefish: A. frenatus only associates with lineage D, whereas A. clarkii lives in the other three lineages.

Counting Nemo: anemonefish Amphiprion ocellaris identify species by number of white bars.

The brilliant colors of coral reef fish have received much research attention. This is well exemplified by anemonefish, which have distinct white bar patterns and inhabit host anemones and defend them as a territory. The 28 described species have between 0 and 3 white bars present, which has been suggested to be important for species recognition. In the present study, we found that Amphiprion ocellaris (a species that displays three white bars) hatched and reared in aquaria, when faced with an intruder fish, attacked their own species more frequently than other species of intruding anemonefish. Additionally, we explicitly tested whether this species could distinguish models with different numbers of bars. For this, 120 individuals of A. ocellaris were presented with four different models (no bars, and 1, 2 and 3 bars) and we compared whether the frequency of aggressive behavior towards the model differed according to the number of bars. The frequency of aggressive behavior toward the 3-bar model was the same as against living A. ocellaris, and was higher than towards any of the other models. We conclude that A. ocellaris use the number of white bars as a cue to identify and attack only competitors that might use the same host. We considered this as an important behavior for efficient host defense.

The ecological function of thyroid hormones.

Thyroid hormones (TH) are central hormonal regulators, orchestrating gene expression and complex biological processes vital for growth and reproduction in variable environments by triggering specific developmental processes in response to external cues. TH serve distinct roles in different species: inducing metamorphosis in amphibians or teleost fishes, governing metabolic processes in mammals, and acting as effectors of seasonality. These multifaceted roles raise questions about the underlying mechanisms of TH action. Recent evidence suggests a shared ecological role of TH across vertebrates, potentially extending to a significant portion of bilaterian species. According to this model, TH ensure that ontogenetic transitions align with environmental conditions, particularly in terms of energy expenditure, helping animals to match their ontogenetic transition with available resources. This alignment spans post-embryonic developmental transitions common to all vertebrates and more subtle adjustments during seasonal changes. The underlying logic of TH function is to synchronize transitions with the environment. This review briefly outlines the fundamental mechanisms of thyroid signalling and shows various ways in which animals use this hormonal system in natural environments. Lastly, we propose a model linking TH signalling, environmental conditions, ontogenetic trajectory and metabolism. This article is part of the theme issue 'Endocrine responses to environmental variation: conceptual approaches and recent developments'.

Henri de Lacaze-Duthiers and the ascidian hypothesis.

In 1830, Cuvier and Geoffroy Saint-Hilaire confronted each other in a famous debate on the unity of the animal kingdom, which permeated the zoology of the 19th century. From that time, a growing number of naturalists attempted to understand the large-scale relationships among animals. And among all the questions, that of the origin of vertebrates was one of the most controversial. Analytical methods based on comparative anatomy, embryology and paleontology were developed to identify convincing homologies that would reveal a logical sequence of events for the evolution of an invertebrate into the first vertebrate. Within this context, several theories have clashed on the question of the identity of the ancestor of vertebrates. Among the proposals, a group of rather discrete organisms, the ascidians, played a central role. Because he had discovered an ascidian with a particularly atypical larval development, the Molgula, Henri de Lacaze-Duthiers, a rigorous and meticulous naturalist, became involved in the ascidian hypothesis. While the visionary mind of Lacaze-Duthiers led him to establish a particularly innovative methodology and the first marine biology station in Europe, at Roscoff, the tailless tadpole of the Molgula prevented him from recognizing the ancestor of vertebrates. This old 19th century story echoes the ever-present questions driving the field of Eco-Evo-Devo.

Anemonefishes: A model system for evolutionary genomics.

Anemonefishes are an iconic group of coral reef fish particularly known for their mutualistic relationship with sea anemones. This mutualism is especially intriguing as it likely prompted the rapid diversification of anemonefish. Understanding the genomic architecture underlying this process has indeed become one of the holy grails of evolutionary research in these fishes. Recently, anemonefishes have also been used as a model system to study the molecular basis of highly complex traits such as color patterning, social sex change, larval dispersal and life span. Extensive genomic resources including several high-quality reference genomes, a linkage map, and various genetic tools have indeed enabled the identification of genomic features controlling some of these fascinating attributes, but also provided insights into the molecular mechanisms underlying adaptive responses to changing environments. Here, we review the latest findings and new avenues of research that have led to this group of fish being regarded as a model for evolutionary genomics.

Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA.

Transcription factors, such as nuclear receptors achieve precise transcriptional regulation by means of a tight and reciprocal communication with DNA, where cooperativity gained by receptor dimerization is added to binding site sequence specificity to expand the range of DNA target gene sequences. To unravel the evolutionary steps in the emergence of DNA selection by steroid receptors (SRs) from monomeric to dimeric palindromic binding sites, we carried out crystallographic, biophysical and phylogenetic studies, focusing on the estrogen-related receptors (ERRs, NR3B) that represent closest relatives of SRs. Our results, showing the structure of the ERR DNA-binding domain bound to a palindromic response element (RE), unveil the molecular mechanisms of ERR dimerization which are imprinted in the protein itself with DNA acting as an allosteric driver by allowing the formation of a novel extended asymmetric dimerization region (KR-box). Phylogenetic analyses suggest that this dimerization asymmetry is an ancestral feature necessary for establishing a strong overall dimerization interface, which was progressively modified in other SRs in the course of evolution.

The multi-level regulation of clownfish metamorphosis by thyroid hormones.

Most marine organisms have a biphasic life cycle during which pelagic larvae transform into radically different juveniles. In vertebrates, the role of thyroid hormones (THs) in triggering this transition is well known, but how the morphological and physiological changes are integrated in a coherent way with the ecological transition remains poorly explored. To gain insight into this question, we performed an integrated analysis of metamorphosis of a marine teleost, the false clownfish (Amphiprion ocellaris). We show how THs coordinate a change in color vision as well as a major metabolic shift in energy production, highlighting how it orchestrates this transformation. By manipulating the activity of liver X regulator (LXR), a major regulator of metabolism, we also identify a tight link between metabolic changes and metamorphosis progression. Strikingly, we observed that these regulations are at play in the wild, explaining how hormones coordinate energy needs with available resources during the life cycle.

The chromosome-scale genome assembly of the yellowtail clownfish Amphiprion clarkii provides insights into the melanic pigmentation of anemonefish.

Anemonefish are an emerging group of model organisms for studying genetic, ecological, evolutionary, and developmental traits of coral reef fish. The yellowtail clownfish Amphiprion clarkii possesses species-specific characteristics such as inter-species co-habitation, high intra-species color variation, no anemone specificity, and a broad geographic distribution, that can increase our understanding of anemonefish evolutionary history, behavioral strategies, fish-anemone symbiosis, and color pattern evolution. Despite its position as an emerging model species, the genome of A. clarkii is yet to be published. Using PacBio long-read sequencing and Hi-C chromatin capture technology, we generated a high-quality chromosome-scale genome assembly initially comprised of 1,840 contigs with an N50 of 1,203,211 bp. These contigs were successfully anchored into 24 chromosomes of 843,582,782 bp and annotated with 25,050 protein-coding genes encompassing 97.0% of conserved actinopterygian genes, making the quality and completeness of this genome the highest among all published anemonefish genomes to date. Transcriptomic analysis identified tissue-specific gene expression patterns, with the brain and optic lobe having the largest number of expressed genes. Further analyses revealed higher copy numbers of erbb3b (a gene involved in melanocyte development) in A. clarkii compared with other anemonefish, thus suggesting a possible link between erbb3b and the natural melanism polymorphism observed in A. clarkii. The publication of this high-quality genome, along with A. clarkii's many unique traits, position this species as an ideal model organism for addressing scientific questions across a range of disciplines.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Colour patterns influence symbiosis and competition in the anemonefish-host anemone symbiosis system.

Colour patterns in fish are often used as an important medium for communication. Anemonefish, characterized by specific patterns of white bars, inhabit host anemones and defend the area around an anemone as their territory. The host anemone is used not only by the anemonefish, but also by other fish species that use anemones as temporary shelters. Anemonefish may be able to identify potential competitors by their colour patterns. We first examined the colour patterns of fish using host anemones inhabited by Amphiprion ocellaris as shelter and compared them with the patterns of fish using surrounding scleractinian corals. There were no fish with bars sheltering in host anemones, although many fish with bars were found in surrounding corals. Next, two fish models, one with white bars and the other with white stripes on a black background, were presented to an A. ocellaris colony. The duration of aggressive behaviour towards the bar model was significantly longer than that towards the stripe model. We conclude that differences in aggressive behaviour by the anemonefish possibly select the colour patterns of cohabiting fish. This study indicates that colour patterns may influence not only intraspecific interactions but also interspecific interactions in coral reef ecosystems.

Transcriptomes of Giant Sea Anemones from Okinawa as a Tool for Understanding Their Phylogeny and Symbiotic Relationships with Anemonefish.

The relationship between anemonefish and sea anemones is one of the most emblematic examples of mutualistic symbiosis in coral reefs. Although this is a textbook example, the major aspects of this symbiosis are still not fully understood in mechanistic terms. Moreover, since studies of this relationship have usually been focused on anemonefish, much less is known about giant sea anemones, their similarities, their phylogenetic relationships, and their differences at the molecular level. Since both partners of the symbiotic relationship are important, we decided to explore this well-known phenomenon from the perspective of giant sea anemones. Here, we report reference transcriptomes for all seven species of giant sea anemones that inhabit fringing reefs of Okinawa (Japan) and serve as hosts for six species of local anemonefish. Transcriptomes were used to investigate their phylogenetic relations, genetic differences and repertoires of nematocyte-specific proteins. Our data support the presence of three distinct groups corresponding to three genera: Entacmaea, Heteractis, and Stichodactyla. The basal position among the three groups belongs to Entacmaea, which was the first to diverge from a common ancestor. While the magnitude of genetic difference between the representatives of Entacmaea and Stichodactyla is large, intra-specific variation within Stichodactyla is much smaller and seems to result from recent speciation events. Our data reconfirms that Heteractis magnifica belongs to the genus Stichodactyla, despite an overall morphological similarity with representatives of the genus Heteractis. The availability of reference transcriptomes will facilitate further research into the fascinating relationship between sea anemones and anemonefish.

Direct development of the catfish pectoral fin: An alternative pectoral fin pattern of teleosts.

BACKGROUND: The study of the teleosts' pectoral fin development touches on many crucial issues of evolutionary biology, from the formation of local adaptations to the tetrapod limbs' origin. Teleosts' pectoral fin is considered a rather developmentally and anatomically conservative structure. It displays larval and adult stages differing in the skeletal and soft tissues' composition. Larva-adult transition proceeds under the thyroid hormone (TH) control that defines pectoral fin ontogeny as an indirect development. However, the outstanding diversity of teleosts allows suggesting the existence of lineage specific developmental patterns. RESULTS: We present a description of the North African catfish, Clarias gariepinus, pectoral fin development. It lacks a clear larval stage and directly develops the adult skeleton with the associated musculature and innervation. Interestingly, the development of catfish pectoral fin appears not to be under the TH dependence. CONCLUSION: This catfish displays a direct pectoral fin developmental trajectory differing from the stereotyped teleost pattern. In the absence of the larval endoskeletal disk and TH control, the catfish's proximal radials arise in a manner somewhat similar to the metapterygial radials in basal actinopterygians and humerus in sarcopterygians. Thus, the catfish fin pattern seems homoplastic, arising by convergence with, or reversion to the ancestral developmental mechanisms.

A chromosome-scale genome assembly of the false clownfish, Amphiprion ocellaris.

The false clownfish Amphiprion ocellaris is a popular fish species and an emerging model organism for studying the ecology, evolution, adaptation, and developmental biology of reef fishes. Despite this, high-quality genomic resources for this species are scarce, hindering advanced genomic analyses. Leveraging the power of PacBio long-read sequencing and Hi-C chromosome conformation capture techniques, we constructed a high-quality chromosome-scale genome assembly for the clownfish A. ocellaris. The initial genome assembly comprised of 1,551 contigs of 861.42 Mb, with an N50 of 863.85 kb. Hi-C scaffolding of the genome resulted in 24 chromosomes containing 856.61 Mb. The genome was annotated with 26,797 protein-coding genes and had 96.62% completeness of conserved actinopterygian genes, making this genome the most complete and high quality among published anemonefish genomes. Transcriptomic analysis identified tissue-specific gene expression patterns, with the brain and optic lobe having the largest number of expressed genes. Further, comparative genomic analysis revealed 91 genome elements conserved only in A. ocellaris and its sister species Amphiprion percula, and not in other anemonefish species. These elements are close to genes that are involved in various nervous system functions and exhibited distinct expression patterns in brain tissue, potentially highlighting the genetic toolkits involved in lineage-specific divergence and behaviors of the clownfish branch. Overall, our study provides the highest quality A. ocellaris genome assembly and annotation to date, whilst also providing a valuable resource for understanding the ecology and evolution of reef fishes.

Small molecules as products of evolution.

The functional evolution of ancient proteins has recently been reconstructed using molecular phylogeny, and the activity of the deduced molecules can be tested. Unfortunately, the world of small molecules escapes such 'resurrection' studies, which rely on ancestral sequence reconstruction. These studies implicitly assume that only the proteins evolve, whereas the small molecules are presumed to be identical now and in the past. Recent evolutionary analysis of biochemical pathways, however, as well as the impressive surge of metabolomics are changing this situation, and it is becoming possible to reconstruct ancient biochemical pathways. We can now begin to infer the chemical structures of key molecules that were present in the past, synthesize those molecules, and test their ability to interact with their cognate (ancient) protein partners. In this essay, we discuss the possibilities offered by these new methodological developments and provide key examples. We also highlight four principles that are important to consider when studying the evolution of small molecules: catalytic promiscuity, metabolic reconfiguration, coevolution and bidirectional interactions. These new developments call for an alliance between organic chemists and evolutionary scientists to investigate the diversity of the chemical building blocks of life, and the evolution of their biosynthetic pathways.

Variation on a theme: pigmentation variants and mutants of anemonefish.

Pigmentation patterning systems are of great interest to understand how changes in developmental mechanisms can lead to a wide variety of patterns. These patterns are often conspicuous, but their origins remain elusive for many marine fish species. Dismantling a biological system allows a better understanding of the required components and the deciphering of how such complex systems are established and function. Valuable information can be obtained from detailed analyses and comparisons of pigmentation patterns of mutants and/or variants from normal patterns. Anemonefishes have been popular marine fish in aquaculture for many years, which has led to the isolation of several mutant lines, and in particular color alterations, that have become very popular in the pet trade. Additionally, scattered information about naturally occurring aberrant anemonefish is available on various websites and image platforms. In this review, the available information on anemonefish color pattern alterations has been gathered and compiled in order to characterize and compare different mutations. With the global picture of anemonefish mutants and variants emerging from this, such as presence or absence of certain phenotypes, information on the patterning system itself can be gained.

Thyroid hormones regulate the formation and environmental plasticity of white bars in clownfishes.

Determining how plasticity of developmental traits responds to environmental conditions is a challenge that must combine evolutionary sciences, ecology, and developmental biology. During metamorphosis, fish alter their morphology and color pattern according to environmental cues. We observed that juvenile clownfish (Amphiprion percula) modulate the developmental timing of their adult white bar formation during metamorphosis depending on the sea anemone species in which they are recruited. We observed an earlier formation of white bars when clownfish developed with Stichodactyla gigantea (Sg) than with Heteractis magnifica (Hm). As these bars, composed of iridophores, form during metamorphosis, we hypothesized that timing of their development may be thyroid hormone (TH) dependent. We treated clownfish larvae with TH and found that white bars developed earlier than in control fish. We further observed higher TH levels, associated with rapid white bar formation, in juveniles recruited in Sg than in Hm, explaining the faster white bar formation. Transcriptomic analysis of Sg recruits revealed higher expression of duox, a dual oxidase implicated in TH production as compared to Hm recruits. Finally, we showed that duox is an essential regulator of iridophore pattern timing in zebrafish. Taken together, our results suggest that TH controls the timing of adult color pattern formation and that shifts in duox expression and TH levels are associated with ecological differences resulting in divergent ontogenetic trajectories in color pattern development.

The real Nemo movie: Description of embryonic development in Amphiprion ocellaris from first division to hatching.

BACKGROUND: Amphiprion ocellaris is one of the rare reef fish species that can be reared in aquaria. It is increasingly used as a model species for Eco-Evo-Devo. Therefore, it is important to have an embryonic development table based on high quality images that will allow for standardized sampling by the scientific community. RESULTS: Here we provide high-resolution time-lapse videos to accompany a detailed description of embryonic development in A ocellaris. We describe a series of developmental stages and we define six broad periods of embryogenesis: zygote, cleavage, blastula, gastrula, segmentation, and organogenesis that we further subdivide into 32 stages. These periods highlight the changing spectrum of major developmental processes that occur during embryonic development. CONCLUSIONS: We provide an easy system for the determination of embryonic stages, enabling the development of A ocellaris as a coral reef fish model species. This work will facilitate evolutionary development studies, in particular studies of the relationship between climate change and developmental trajectories in the context of coral reefs. Thanks to its lifestyle, complex behavior, and ecology, A ocellaris will undoubtedly become a very attractive model in a wide range of biological fields.

A structural signature motif enlightens the origin and diversification of nuclear receptors.

Nuclear receptors are ligand-activated transcription factors that modulate gene regulatory networks from embryonic development to adult physiology and thus represent major targets for clinical interventions in many diseases. Most nuclear receptors function either as homodimers or as heterodimers. The dimerization is crucial for gene regulation by nuclear receptors, by extending the repertoire of binding sites in the promoters or the enhancers of target genes via combinatorial interactions. Here, we focused our attention on an unusual structural variation of the α-helix, called π-turn that is present in helix H7 of the ligand-binding domain of RXR and HNF4. By tracing back the complex evolutionary history of the π-turn, we demonstrate that it was present ancestrally and then independently lost in several nuclear receptor lineages. Importantly, the evolutionary history of the π-turn motif is parallel to the evolutionary diversification of the nuclear receptor dimerization ability from ancestral homodimers to derived heterodimers. We then carried out structural and biophysical analyses, in particular through point mutation studies of key RXR signature residues and showed that this motif plays a critical role in the network of interactions stabilizing homodimers. We further showed that the π-turn was instrumental in allowing a flexible heterodimeric interface of RXR in order to accommodate multiple interfaces with numerous partners and critical for the emergence of high affinity receptors. Altogether, our work allows to identify a functional role for the π-turn in oligomerization of nuclear receptors and reveals how this motif is linked to the emergence of a critical biological function. We conclude that the π-turn can be viewed as a structural exaptation that has contributed to enlarging the functional repertoire of nuclear receptors.