The scattered information regarding wild and commercial pigmentation mutants of anemonefishes has been gathered in this work, and compiled and categorized into two classes, encompassing either chromatophore imbalances (class I) or patterning irregularities (class II).
What global picture emerges from these data? Interestingly, although several classes of mutant phenotypes can be identified, there are several potential phenotypes that do not seem to exist, predominantly the complete absence of chromatophore subtypes (lack of xanthophores or lack of all chromatophores). In order to learn something about pigmentation pattern formation, biological processes that could lead to the observed mutant phenotypes must be taken into consideration and investigated in detail. Even though much more data, especially experimental data, are needed to perform these analyses, we will try, in the following discussion, to link selected anemonefish mutant phenotypes to potential processes affected.
What can we learn about color pattern formation from pigmentation mutants?
Classification of pigmentation phenotypes in zebrafish and medaka has allowed researchers to clarify which biological process(es) might be affected by the underlying mutation. As pigmentation heavily relies on chromatophores and their spatial arrangement, the neural crest development is of particular interest. Key developmental processes are (1) specification, (2) proliferation, (3) differentiation, (4) survival, and (5) patterning [23]. Disruptions in any of these have the potential to alter pigmentation patterns.
Chromatophore specification
Defects during specification are likely present if chromatophore numbers are strongly reduced, but the remaining cells appear normal and in the correct position.
Mutations related to “Naked” phenotypes (highly reduced white barring, Fig. 4B–G) are most likely caused by genes responsible for iridophore development, in particular specification. As mentioned above, the reduction of iridophores will indirectly reduce the number of melanophores that form the edge of the white bars, but melanophore distribution remains normal in fin and body coloration. In zebrafish, several genes have been linked with iridophore specification and they can serve as an entry point to investigate “Naked”-typed mutants. For example, Foxd3 expression is required for neural crest derivatives to develop into iridophores [29]. Leucocyte tyrosine kinase (ltk) controls iridophore establishment, proliferation, and survival, and is associated with at least two mutants, “shady” (lack of iridophores) and “moonstone” (ectopic iridophores) [13]. Endothelin receptor signaling is crucial for larval iridophores highlighted by the importance of genes encoding Edn3b (Endothelin 3b) and Ednr3b (Endothelin receptor b1a) [28].
Apart from iridophores, other chromatophore subtypes might be also affected, as demonstrated in zebrafish (e.g.: the “nacre” mutant does not possess melanophores) and guppies (e.g.: the “blue” mutant lacks xanthophores). However, in anemonefish specification defects of either melanophores or xanthophores have not been discovered yet, neither in the wild (apart for the “Golden Clownfish”, Fig. 3G), nor in aquaculture. Even though some retail mutant strains are nearly or completely white (A. ocellaris “Wyoming White”, Fig. 4N, A. percula “Platinum/Maine Blizzard”, Fig. 4M) or completely black (A. ocellaris “Midnight”, Fig. 4F) as adults, larvae and juveniles of these lines display an orange body coloration that is slowly replaced by a different color during maturation (own personal observation, correspondence with aquaculture companies). The face and in particular the region around the mouth is usually the last to change color as shown in the “Midnight” mutant (Fig. 4F). The “Golden Clownfish” (Fig. 3G) is the only known individual that appears to lack both iridophores and melanophores, leaving only xanthophores to color the entire body uniform yellow.
All three chromatophore subtypes are affected when genes required for normal development of neural crest derivatives are mutated, such as mutations of Sox10 (SRY-related HMG-box 10). The corresponding zebrafish mutant is called “colorless” [23] and the same gene is also required for chromatophore development in guppies [12]. To the best of our knowledge, no anemonefish lacking all three chromatophore subtypes has yet been found. Neural crest derivatives do not only specify chromatophore subtypes, but also craniofacial cartilage, peripheral and enteric neurons as well as glia [4, 22]. It is possible that in anemonefish mutations that affect these additional cell types are lethal and therefore have not been identified so far.
Of course, it is difficult to go further with analyses of the existing mutants without more detailed information. But from the data available so far, very interesting and focused questions emerge: Do xanthophores and melanophores in anemonefish or rather their precursors have other functions that if disrupted lead to highly increased mortality? Alternatively, it may be that the specification process for these cells can take alternative routes, so even if one gene is knocked out, others can replace it so that the precursor is still established. But why should this situation be different for iridophores? All these questions are awaiting molecular and developmental characterization of these specification mutants.
Chromatophore proliferation
Proliferation defects can also be indicated by a strongly reduced number of chromatophores as well as normal and correct positions of the remaining chromatophores.
Many retail lines display a pronounced shift towards either partially or completely white or black morphotypes. However, this does not necessarily mean that the establishment of the xanthophore lineage is negatively affected. For example, larvae and juveniles of many of these lines (such as “Darwin Black”, “Black Storm”, “Snow Storm”, and “Wyoming White”) usually display an orange body coloration that is slowly replaced by black color during maturation. This suggests that initial xanthophore specification and proliferation processes are unaffected, but their subsequent fate remains to be analyzed. Possible scenarios include (1) melanophores (or iridophores) replacing xanthophores; (2) xanthophores changing chromatophore subtype identity, or (3) xanthophores remaining present but becoming covered by other chromatophores. For example, anemonefish skin chromatophores are distributed within the epidermis as well as the dermis [55, 56] and it is possible that changes in the top layers cover the coloration of the deeper layers. In this hypothesis, it is assumed that xanthophores remain present in a deeper epidermal layer, while melanophores (or iridophores) spread in a more upper layer and therefore overlay the orange color. Regardless of the exact mechanism, proliferative processes of melanophores (or iridophores, respectively) are enhanced above normal levels during juvenile color maturation.
Differentiation processes
Alterations of cell differentiation processes can include a variety of phenotypes, such as reduced or altered pigmentation or abnormal cell morphology.
A first example for a differentiation defect is the “Zombie” mutation (Fig. 4H), which is most likely caused by the lack of melanin (albinism). In albinism, melanophores are present in their normal arrangement and distribution but they are de-pigmented, either completely lacking melanin or displaying highly reduced melanin levels [39, 63]. Since melanophores are present in these mutants, regulatory genes, such as Mitfa (microphthalmia transcription factor a) or kita (receptor tyrosine kinase a), are unlikely to be affected in these mutants. Genetic mutations in a gene involved in melanogenesis can potentially result in reduced or absent melanin synthesis leading to reduced pigmentation levels. The biosynthetic pathway during which L-tyrosine is converted to melanin through several enzyme catalyzed steps involves many genes, such as MC1R (melanocortin 1 receptor), tyr (tyrosinase), tyrp1 (tyrosinase-related protein-1), and dct/tyrp2 (dopachrome tautomerase). The most well documented example is MCR1, which contributes to melanin-related polymorphisms in several animal species, including the guppy Poecilia reticulata [61], zebrafish [51], and various vertebrates [19]. Another potential candidate is oca2 (oculocutaneous albinism II). When its function is disrupted, the first step of melanin synthesis, the conversion of L-tyrosine to L-DOPA, is impaired, and this situation is responsible for the evolution of albinism in the cavefish Astyanax mexicanus [27]. Any of the above-mentioned genes might be responsible for the “Zombie” mutants in anemonefish. Interestingly, the first “Zombie” mutant first arose from a pair of “Black/Darwin” A. ocellaris (Fig. 4A). Juvenile “Zombie” mutants display a bright yellow-orange body coloration with white bars devoid of the black edge and have red eyes. Later on, individuals turn a darker shade of orange-reddish to reddish-brown. This suggests that some melanin is produced and accumulates, resulting in the darker shades of mature individuals. Because the original “Zombie” arose from completely black parents, melanophores cover the entire body and the little melanin that is produced is evenly distributed.
Another example of altered pigment cell differentiation is the icy blue, iridescent hue (instead of the normal white coloration) that is usually more prominent near black coloration. This altered pigmentation feature is most often seen in black and white morphotypes of A. ocellaris mutant lines, such as “Black Frostbite” or “Black Snowflake” (see also https://reefbuilders.com/2018/03/05/blue-clownfish/). It is noteworthy that some species of anemonefish such as A. chrysopterus, A. frenatus, A. melanopus, and P. biaculeatus, show an increased bluish appearance at the black border as part of their normal pigmentation patterns. This phenotype is most likely linked to the arrangement and organization of guanine platelets and crystals within the iridophores. As mentioned above, an iridescent color can be observed when crystals are aggregated in platelets that are precisely organized, while they appear whitish when the crystal platelets are less organized as light is scattered in various directions [15, 59]. Guanine platelet formation requires gmps (guanine monophosphate synthetase) expression [42] and a mutation in this gene could potentially alter platelet formation. However, since the blue-iridescent hue of iridophores usually appears near the black border, it is also possible that the presence of melanophores is partially responsible for guanine crystals to be orientated differently. Interestingly, the existence of two different subtypes of iridophores has been shown recently in zebrafish [17] and the authors suggest that the presence of melanophores might affect the patterning of both iridophore subtypes.
Pigmentation patterning
Patterning mechanisms are likely affected if a subset of the pattern is absent or altered in any way (e.g.: position, extra elements). In general, these changes are very hard to predict and will most likely affect tissues other than the neural crest.
In anemonefishes, major patterning factors concern the anterior–posterior succession of white bars. Both the position as well as the sequence of white bar appearance appears highly constrained. All species of anemonefish that possess white bars have them at the same position: (1) the transition of head to body; (2) the middle of the body, and (3) the caudal peduncle/tail. This suggests that the same morphological landmarks are used to establish the exact position of the bars. For the white body bar, it has been suggested that the transition between anterior spines and posterior soft rays of the dorsal fin acts as a pre-positioning cue [58]. More specifically, an indentation (made by longer anterior spines and shorter posterior spines) is more pronounced in species that have two or three white bars and less obvious in species with zero or one bar [58]. In addition to the exact position of the white bar, their developmental appearance is also highly conserved and constrained, as mentioned above. No species exhibits a single bar on the body or tail only. So far, only one wild-caught fish has been found that exhibits a disruption in this normal anterior–posterior patterning (Fig. 3X). This specimen lacked the head bar but showed normal formation of the body and tail bars. As there usually is an anterior to posterior sequence of bar acquisition during larval/juvenile development, this specimen is very fascinating, but unfortunately, its fate is unknown. Therefore, it is unclear if the underlying mechanisms are inheritable, and the molecular and cellular mechanisms responsible for the anterior to posterior sequence of bar appearance/formation remain to be analyzed.
Pigmentation research on anemonefishes offers the unique opportunity to study boundary formation—with melanophores forming a distinct border between the other two chromatophore subtypes, which is much harder to investigate in zebrafish or medaka (zebrafish: differently colored stripes, medaka: uniform color). It can therefore be assumed that novel processes will be revealed from such future analyses. Like the underlying mechanisms for anterior–posterior patterning, the genetic cause(s) for mutations that result in abnormal bar shape are rather hard to predict. The failure of different subtypes of chromatophores to communicate and interact could be responsible for altered boundary formation. Indeed, preliminary data suggests that melanophores restrict iridophore expansion (our own observation). Therefore, if melanophore regeneration and migration abilities are impaired, this could potentially lead to altered bar shapes. As outlined above, differences can be observed between several types of bar shape mutations. “Picasso”-typed mutations (Fig. 4I–L) are the most widespread and have been found in at least eight different species of anemonefish (A. percula, A. ocellaris, A. clarkii, A. bicinctus, A. sebae, A. frenatus/A. melanopus, A. polymnus, and P. biaculeatus). Moreover, the striking similarities between heterozygous and homozygous phenotypic appearances (compare Fig. 4J/4 M with 4 K/4 N with 4L/4O) in three species (A. percula, A. ocellaris, P. biaculeatus) suggests that the underlying genetic mechanisms are very similar, if not the same, while phenotypic appearance remains highly variable. Comparative developmental analyses of different species would be helpful to investigate when color alterations first appear and if they are similar in different species. Furthermore, genetic analyses of all species showing “Picasso”-typed mutations would show if the same genes are indeed affected.
Preliminary research on “Snowflake” A. ocellaris (Fig. 4P) suggest that a local decrease of melanophores forming the black edge might enable the iridophores to expand into neighboring areas, leading to the jagged black outline of this mutation (own observation).
Ecological and evolutionary implications of color pattern variations
The ecological functions of color patterns in anemonefishes are still unclear, probably because experimental studies are scarce. However, it has been hypothesized that color might play a role in various functions, such as (1) concealment and camouflage either from predators [36, 58] or from resident fish in the case of juveniles [6], (2) species recognition and co-existence [58], and (3) individual identification [8]. Alteration of color pattern via mutation might then affect the adaptive value of the color pattern trait, and hence the fitness of the individual. Indeed, some color pattern mutations observed in the aquarium trade are never or very rarely observed in the wild, for example almost completely white morphotypes or individuals with no bands in species that usually have bands. This suggests that mutations which result in drastic color pattern alterations might have a negative effect on the survival of individuals in the wild and are therefore negatively selected against. However, other mutations are more widespread (like melanism, and “Picasso”-typed bar shape alterations), and individuals survive long enough to reach reproductive positions. This indicates that these mutations have no or negligible negative effects on survival, as the ascension to breeding positions is a lengthy process as juveniles go up the social hierarchy [7]. In the aquarium trade, A. ocellaris is by far the anemonefish species that exhibits the most color mutations, followed by P. biaculeatus (Additional file1: Table S1). In the wild, most mutant individuals are P. biaculeatus or A. perideraion, and have been noted particularly from within the Coral Triangle area. Ecological field studies within the Ryukyu Archipelago revealed relatively high rates of abnormal coloration in A. perideraion (1.8%) and A. sandaracinos (2.56%), but negligible rates for the remaining four anemonefish species present in this region (A. clarkii, A. frenatus, A. ocellaris, and A. polymnus) (Kina Hayashi, unpublished observation). From a limited Internet search using the photo sharing platform flickr (https://www.flickr.com/explore), a total of 36,195 pictures that were tagged as “clownfish” were screened for images of wild mutant individuals. Only approximately half of those images displayed natural marine environments, from which 34 aberrant anemonefish images were retrieved (0.18%). This figure is an only gross estimate and most likely represents a minimum of wild mutant anemonefishes. On the other hand, it may be that wild anemonefish variants are more noticeable and recreational divers might take more photographs of unusual individuals, and clearly more data from field observations are needed to better establish the true rates of abnormal coloration of anemonefish. As well, wild-caught specimens with aberrant color patterns are definitively targeted by export companies to be sold to aquaculture companies or private collectors [37].
From these observations, it can be concluded that most variants are rare and therefore selected against, implying that variations do not provide any advantages. It is possible that even though mutant individuals may reach breeding positions, the underlying mutation is usually not transmitted to an increased number of aberrant juveniles, that in turn would join or establish colonies. One reason could be that juveniles with color alterations are less successful in joining an existing colony precisely because they exhibit a different color pattern. The investigation of the ecological function(s) of color patterns would greatly benefit from experimental studies using mutants, which could provide new insights on this poorly understood aspect of anemonefish ecology. For example, in mesocosm experiments it would be interesting to study how juveniles with different white bar patterns are differentially affected during the recruitment phase when joining an existing colony. Moreover, the effect of changes in bar patterns on the aggressiveness of conspecifics could also be studied. These types of analyses are currently underway in our laboratory.
Finally, it should be noted that some polymorphic variations in bar shape are observed more frequently in the wild but are not regarded to be the results of mutations. These include, for example, (1) the shape of the middle bar in A. polymnus, (2) the head bar in A. nigripes which can be joined dorsally or display a gap, and (3) the tendency of all three bars to disappear in a ventral to dorsal fashion in older individuals of P. biaculeatus and A. ocellaris. The last case may be linked to senescence related degeneration processes.
Research has suggested that approximately half of all coral reef fish species have evolved in the last 5 Myr [3]. While much of this massive radiation of new species is thought to have been driven by biogeographic conditions, colors and the patterns of fish are also thought to have played important roles [48], perhaps via stabilizing species boundaries [3]. Recent phylogenetic analyses of Amphiprioninae with molecular clock analyses show that a large majority of anemonefish species have also evolved within the last 5 Myr, likely due to biogeography combined with ecological speciation related to host-anemone associations [30, 32]. This Amphiprioninae phylogenetic framework combined with recent phylogenetic data on anemones [62] and the analyses of coloration and patterning as suggested in this review promise to open exciting new avenues of research that will allow a better understanding of the evolution of anemonefish pigmentation diversity.
