Anemonefishes: A model system for evolutionary genomics

Abstract

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.

Keywords: Amphiprion; adaptive radiation; chromosome-scale assembly; clownfish; genome; pigmentation; proteomics; transcriptomics.

Figure 1.. Overview of the “omics” technologies used in anemonefish research.

The green circle represents metabolomics, orange -proteomics, blue -transcriptomics, and pink -genomics. Colored arrows indicate interactions between the metabolome, proteome, transcriptome, and genome and how they affect each other. Circle sizes illustrate estimated complexity (adapted from Grimm et al. 2021). Number of publications using “omics” tools were retrieved from the Web of Knowledge ( https://apps.webofknowledge.com/) and plotted according to year of publication. Since the early 2000’s when the first studies investigating gene expression and technological advancement of various molecular sequencing platforms, the application of “omics” tools has increased steadily and led to the achievement of milestones such as the assembly of one of the most contiguous chromosome-scale fish genomes and the successful use of CRISPR/Cas9 gene editing in a reef fish (as shown by the light areas in the plot). Keywords used to determine these studies were separated into independent variables (or) within two categories donated by (and): “gene expression or genome or transcriptome or proteome or genomics or transcriptomics or proteomics or omics” and “clownfish or anemonefish or Amphiprion”.

Figure 2.. Mutualism with sea anemones triggered the adaptive radiation of anemonefish.

a) Phylogeny of anemonefishes based on the 20 most informative genes (adapted from Marcionetti et al. 2022). Geographical distributions (light blue: NWI – North-Western Indian Ocean, dark blue: WI – Western Indian Ocean, green: CIP –Central Indo-Pacific Ocean, orange: CP – Central Pacific Ocean, yellow: SWP – South-Western Pacific Ocean, red: P – Polynesian Ocean), sea anemone hosts, and phenotypes are shown for each species. Asterisk denotes a recent revision of anemonefish phylogenetic data that suggests Premnas biaculeatus should be recognized as Amphiprion. Lastly, DNA symbol is shown next to the species for which genomes have been sequenced. b) The iconic false clownfish Amphiprion ocellaris. c) The white bonnet anemonefish Amphiprion leucokranos is a naturally occurring hybrid species found in the WI and CIP regions. d) Clownfish lay hundreds of eggs on the substrate near their host anemone. Pictures taken by Pascal Kobeh.

Figure 3.. Advances in genomics of anemonefish.

a) A comparison of genome contiguity for the three anemonefish chromosome-scale genomes (the false clownfish Amphiprion ocellaris, the orange clownfish Amphiprion percula, and the yellowtail clownfish Amphiprion clarkii) and 26 other previously published chromosome-scale fish genomes assemblies until 2019 (adapted from Lehman et al. 2019 and Hotaling et al. 2019). Data points are color-coded by order. b) Chromatin contact mapping takes advantage of the inverse relationship between proximity of nuclear DNA and genomic distance thus allowing contigs to be clustered into chromosomal groups. Here, the Hi-C heatmap of interactions between pairs of chromosomal loci (chr01-chr24) throughout the A. clarkii genome is shown. Interactions were drawn based on the chromatin interaction frequencies between pairs of 100 kb genomic regions (as determine by Hi-C). Darker red cells indicate stronger and more frequent interactions, which in turn imply that the two sequences are spatially close. Close-ups on the right show an overview of features revealed by Hi-C maps. Top squares show the long-range contact pattern of a locus (left) and its nuclear subcompartments (right). Middle squares show enhanced contact frequency along the diagonal (left) which indicate the presence of small domains of condensed chromatin (right). Bottom squares show peaks in the contact map (left) and the presence of loops that lie at domain boundaries and bind CTCF (right) (adapted from Rao et al. 2014). c) Availability of high-quality chromosome scale assemblies allow for large-scale genomic comparisons. Here, a dual synteny plot between all 24 chromosomes from A. ocellaris and A. percula shows conserved sequences within chromosomes of both species. Chromosomal rearrangements such as translocations and inversions are shown as red ribbons, whereas blue ribbons represent unchanged regions. Chromosomes in both species have been designated based on their size following and orderly arrangement from the largest to the smallest.

Figure 4.. The mutualistic relationship between clownfish (CF) and sea anemones (SA) has been a long-standing question in anemonefish research.

Two conflicting hypotheses have been proposed to explain how anemonefish are able to live safely in their host: 1) CF acquire antigens of the SA mucus that protects them from being stung, and 2) CF produce their own protective mucus, which either prevents nematocyst discharge by the host or protects the fish from the consequences of the sting. Particularly, N-acetylneuraminic acid (Neu5Ac) might have a critical role in the chemical recognition of the host.

Figure 5.. New insights into the processes generating complex pigmentation patterns in reef fish.

a) Color patterns in anemonefish can vary greatly depending on their ecology, development, and evolution (adapted from Refs. 15, 178). White bars could be necessary for species recognition and could be adaptive for camouflage or even used as an aposematic signal. Pigmentation polymorphisms have also been observed in Amphiprion percula living in Heteractis or Stichodactyla anemones: 1) juveniles in Heteractis exhibit a delayed white bar formation and 2) adults in Stichodactyla show higher melanism. The three white bars arise sequentially from anterior to posterior body parts during ontogenesis whereas during evolution, bars are lost in the opposite sequence of ontogenesis (from the posterior to anterior region). b) Natural melanism polymorphism observed in Amphiprion clarkii (adapted from Ref. 36). c) Examples of Amphiprion ocellaris color mutants available from aquaculture companies: naked phenotypes (“Naked” and “Extreme Misbar”), phenotypes with extra white markings (“Gladiator/Da Vinci”) to an almost nearly complete white colored body (“Wyoming White”), melanic phenotypes (“Black/Darwin” and “Domino”), and phenotypes with irregular patterning (“Snowflake” and the Premnas biaculeatus “Lightning”) (adapted from Ref. 16).