The symbiosis between clownfishes (or anemonefishes) and their host sea anemones ranks among the most recognizable animal interactions on the planet. Found on coral reef habitats across the Indian and Pacific Oceans, 28 recognized species of clownfishes adaptively radiated from a common ancestor to live obligately with only 10 nominal species of host sea anemones. Are the host sea anemones truly less diverse than clownfishes? Did the symbiosis with clownfishes trigger a reciprocal co-evolutionary response to the mutualism? To address these questions, we combined fine- and broad-scale biogeographic sampling with multiple independent genomic datasets for the bubble-tip sea anemone, Entacmaea quadricolor-the most common clownfish host anemone throughout the Indo-West Pacific. Fine-scale sampling and restriction site associated DNA sequencing (RADseq) throughout the Japanese Archipelago revealed three highly divergent cryptic species: two of which co-occur throughout the Ryukyu Islands and can be differentiated by the clownfish species they host. Remarkably, broader biogeographic sampling and bait-capture sequencing reveals that this pattern is not simply the result of local ecological processes unique to Japan, but part of a deeper evolutionary signal where some species of E. quadricolor serve as host to the generalist clownfish species Amphiprion clarkii and others serve as host to the specialist clownfish A. frenatus. In total, we delimit six cryptic species in E. quadricolor that have diversified within the last five million years. The rapid speciation of E. quadricolor combined with functional ecological and phenotypic differentiation supports the hypothesis that this diversification is an evolutionary response to mutualism with clownfishes. Clownfishes are not merely settling in locally available hosts but recruiting to specialized host lineages with which they have co-evolved. These findings have important implications for understanding how the clownfish-sea anemone symbiosis has evolved and will shape future research agendas on this iconic model system.

The Bermudan Archipelago supports a high-latitude subtropical coral reef ecosystem similar to 1500 km from the nearest major reef system in the Tropical Western Atlantic (TWA) and Greater Caribbean. Although isolated, Bermuda has low rates of marine endemism due to regular long-distance dispersal from the TWA via the Gulf Stream Current. Nevertheless, Bermuda's coral reef biodiversity is a reduced subset of that in the Greater Caribbean, indicating that its isolation does limit some species from dispersing or establishing persistent populations. The Alpheus armatus Rathbun, 1901 species complex is a clade of five pistol snapping shrimp species that form obligate symbioses with sea anemones from the TWA and Greater Caribbean. Although their host anemones are abundant, no members of this complex are known from Bermuda. We provide the first report of the A. armatus species complex from Bermudan coral reefs. Using molecular approaches, we identified our observed individual as Alpheus immaculatus Knowlton and Keller, 1983. Phylogenetic and species delimitation analyses indicate that our A. immaculatus sample from Bermuda does not represent a cryptic lineage nor genetically differentiated population. Instead, we interpret this species to have arrived recently as a chance long-distance dispersal event. No other A. immaculatus individuals were observed, suggesting this species has not been able to establish a long-term population in Bermuda.

Reduced representation sequencing is a powerful population genomics method but is often limited in anthozoans due to the challenges in interpreting mixed host-dinoflagellate data when not using reference genomes. To circumvent endosymbiont contamination, we menthol bleached Condylactis gigantea sea anemones and assembled an aposymbiotic reference sample. Quantitative PCR detected only trace amounts of endosymbiont presence, supporting the efficacy of our bleaching approach. With this, we then reexamined a previously published holobiont ddRADseq dataset to test the hypothesis that reference sample information would eliminate endosymbiont loci and significantly change prior results. Incorporating the aposymbiotic reference sample did not substantively change the results of the prior study. We demonstrate that bleaching a reference sample for reduced representation sequencing may not be justified because of the marginal difference in results. This work supports a recent hypothesis that ddRADseq data assembly pipelines act as de facto endosymbiont filtering steps for symbiotic anthozoans.

Abstract Understanding how symbiosis generates biodiversity requires comprehensive insight into the underlying ecology to reveal broad evolutionary patterns, and the clownfish-anemone symbiosis serves as an excellent model. Recently, a new paradigm revealed sea anemone host associations of adult clownfish drove convergent evolution of host specialists and generalists. An alternative study downplayed host specificity in explaining clownfish evolution due partly to an incongruence between the behavior, biology, and host specificity of a single species— Amphiprion polymnus . Previously classified as a host specialist, we revisit the host specificity and ecology of A. polymnus by studying its host associations, behavioral, and dietary ecology compared to a co-occurring host generalist and specialist. By incorporating n = 358 observations, we find A. polymnus associates with five sea anemone species, including a newly documented host, and four at the adult life stage. We show that A. polymnus swim consistently far from their hosts and have broad dietary niches, an ecology that aligns with generalists. Cumulatively, our data indicate A. polymnus is a host generalist, and not a specialist as previously classified. Our findings serve to reconcile previously conflicting studies about A. polymnus , and reinforce the hypothesis that host is the key ecological variable for understanding clownfish evolution. Suitable subject areas behaviour, ecology, evolution

Signals mediate a range of behavioural interactions with important fitness implications within and between species. However, compared to our knowledge about how signals convey information and remain reliable within species, much less is known for interspecific signalling. One interspecies interaction mediated by signals are cleaning mutualisms in which cleaner shrimp remove parasites from their reef fish clients. The Pederson cleaner shrimp, Ancylomenes pedersoni, signals its intent to clean clients by whipping its long, white antennae, which is usually followed by cleaning. Based on the assumption that intent to clean is driven by hunger, we hypothesized that shrimp advertise willingness to clean based on their own hunger level, with hungrier shrimp being more willing to signal (and clean) than satiated ones. Here, we present support for this hypothesis from both the field and laboratory. First, we annotated field videos collected in Cura & ccedil;ao and Honduras, and found that the likelihood of antennae whipping and cleaning increased as time since last clean increased (with more time passing presumably leading to the shrimp being hungrier). In the laboratory, we manipulated the hunger levels of captive A. pedersoni and exposed them to a visual stimulus of a client fish. We found that the proportion of interactions in which shrimp antennae-whipped and cleaned was significantly greater when shrimp were hungry, compared to when satiated. Interestingly, we also found that signal honesty, how often shrimp followed signalling with cleaning, was less variable in hungry versus satiated shrimp. These results indicate that hunger level influences the signalling and cleaning behaviour of cleaner shrimp, revealing a mechanism underlying the information content of cleaning advertisement signals in A. pedersoni and suggesting a link between hunger level and signal honesty. Overall, this study provides insight into the interspecies signalling that maintains an important mutualistic relationship. (c) 2026 The Association for the Study of Animal Behaviour. Published by Elsevier Ltd. All rights are reserved, including those for text and data mining, AI training, and similar technologies.

Anti-predator coloration strategies vary widely from conspicuous to cryptic, and everything between. Coloration serves multiple functions, and determining the selective pressures that drive this diversity is a long-standing mystery. Clownfishes display enormous inter- and intra-specific variation in coloration without the influence of sexual selection, providing an excellent system in which to explore these drivers in closely related and sympatric species. Across clownfishes, three color-pattern phenotypes have evolved convergently with host anemone use, implying adaptive functions linked to the host’s physical and chemical properties. Here we conducted a comprehensive study to infer the ultimate functions of clownfish color patterns. By integrating multiple levels of biological organization, including behavior, visual ecology, dietary niche, and microbiome, our data overwhelmingly indicate that specialist and generalist clownfish color patterns have different underlying ecologies and adaptive functions. Specialist clownfishes background match their anemones, remain near hosts, and have specialized diets and microbiomes. Generalists are highly contrasting with their anemones, remain far from hosts, and have variable diets and microbiomes. Taken together, specialist color patterns align with functional expectations of camouflage while generalists align with aposematic or disruptive coloration. Our study provides novel insights into how ecological conditions shape the evolution of multiple anti-predator coloration strategies. ### Competing Interest Statement The authors have declared no competing interest. U.S. National Science Foundation, DEB-1934274, PRFB-2305953

Sea anemones form complex holobiont communities that include not only microorganisms (microbiota) but also metazoans, such as anemonefish. Previous studies have revealed the significant impacts of bleaching events on anemone microbiota and on anemonefish physiology. However, the impacts of bleaching on anemonefish microbiota remain understudied, despite the potential for microbial dysbiosis to favor the emergence of pathogens and opportunistic microbes which could be detrimental for anemonefish. As a consequence, we sampled microbiota in situ from healthy and bleached anemones (Radianthus magnifica) and their associated anemonefish (Amphiprion maohiensis) during the 2019 bleaching event in the lagoon of Mo’orea, French Polynesia. Metabarcoding was used to examine the diversity of Symbiodiniaceae and bacterial communities. Our results revealed significant differences in the assemblages of Symbiodiniaceae and bacteria between healthy and bleached anemones. Specifically, the alpha diversity of these microbial communities was higher in bleached anemones, suggesting microbial dysbiosis and an increase in opportunistic taxa. In contrast, no differences were observed in the bacterial assemblages of anemonefish associated with healthy and bleached anemones. Despite this lack of correlation between anemonefish microbiota and anemone bleaching status, we found that anemonefish living within the same anemone had nevertheless similar bacterial assemblages. As a consequence, we hypothesized that anemone bleaching does not significantly impact the microbiota of anemonefish, and that direct skin-to-skin contact among anemonefish is the primary factor influencing their microbiota composition.

The symbiosis between clownfish and giant tropical sea anemones (Order Actiniaria) is one of the most iconic on the planet. Distributed on tropical reefs, 28 species of clownfishes form obligate mutualistic relationships with 10 nominal species of venomous sea anemones. Our understanding of the symbiosis is limited by the fact that most research has been focused on the clownfishes. Chromosome-scale reference genomes are available for all clownfish species, yet only short reads-based reference genomes are available for five species of host sea anemones. Recent studies have shown that the clownfish-hosting sea anemones belong to three distinct clades of sea anemones that have evolved symbiosis with clownfishes independently. Here we present the first high-quality long-read assemblies for three species of clownfish-hosting sea anemones belonging to each of these clades: Entacmaea quadricolor, Stichodactyla haddoni, and Radianthus doreensis. PacBio HiFi sequencing yielded 1,597,562, 3,101,773, and 1,918,148 million reads for E. quadricolor, S. haddoni, and R. doreensis, respectively. All three assemblies were highly contiguous and complete with N50 values above 4 Mb and BUSCO completeness above 95% on the Metazoa dataset. Genome structural annotation with BRAKER3 predicted 20,454, 18,948, and 17,056 protein-coding genes in E. quadricolor, S. haddoni, and R. doreensis genome, respectively. These new resources will form the basis of comparative genomic analyses that will allow us to deepen our understanding of this mutualism from the host perspective.

The symbiosis between clownfishes (or anemonefishes) and their host sea anemones ranks among the most recognizable animal interactions on the planet. Found on coral reef habitats across the Indian and Pacific Oceans, 28 recognized species of clownfishes adaptively radiated from a common ancestor to live obligately with only 10 nominal species of host sea anemones. Are the host sea anemones truly less diverse than clownfishes? Did the symbiosis with clownfishes trigger a reciprocal adaptive radiation in sea anemones, or minimally, a co-evolutionary response to the mutualism? To address these questions, we combined fine- and broad-scale biogeographic sampling with multiple independent genomic datasets for the bubble-tip sea anemone, Entacmaea quadricolor , the most common clownfish host anemone throughout the Indo-West Pacific. Fine-scale sampling and restriction site associated DNA sequencing (RADseq) throughout the Japanese Archipelago revealed three highly divergent cryptic species: two of which co-occur throughout the Ryukyu Islands and can be differentiated by the clownfish species they host. Remarkably, broader biogeographic sampling and bait-capture sequencing reveals that this pattern is not simply the result of local ecological processes unique to Japan, but part of a deeper evolutionary signal where some species of E. quadricolor serve as host to the generalist clownfish species Amphiprion clarkii and others serve as host to the specialist clownfish A. frenatus . In total, we delimit at least five cryptic species in E. quadricolor that have diversified within the last five million years. The rapid diversification of E. quadricolor combined with functional ecological and phenotypic differentiation supports the hypothesis that this may represent an adaptive radiation in response to mutualism with clownfishes. Our data indicate that clownfishes are not merely settling in locally available hosts but recruiting to specialized host lineages with which they have co-evolved. These findings have important implications for understanding how the clownfish-sea anemone symbiosis has evolved and will shape future research agendas on this iconic model system. ### Competing Interest Statement The authors have declared no competing interest.

Recent genomic analyses have highlighted the prevalence of speciation with gene flow in many taxa and have underscored the importance of accounting for these reticulate evolutionary processes when constructing species trees and generating parameter estimates. This is especially important for deepening our understanding of speciation in the sea where fast moving ocean currents, expanses of deep water, and periodic episodes of sea level rise and fall act as soft and temporary allopatric barriers that facilitate both divergence and secondary contact. Under these conditions, gene flow is not expected to cease completely while contemporary distributions are expected to differ from historical ones. Here we conduct range-wide sampling for Pederson’s cleaner shrimp (Ancylomenes pedersoni), a species complex from the Greater Caribbean that contains three clearly delimited mitochondrial lineages with both allopatric and sympatric distributions. Using mtDNA barcodes and a genomic ddRADseq approach, we combine classic phylogenetic analyses with extensive topology testing and demographic modeling (10 site frequency replicates x 45 evolutionary models x 50 model simulations/replicate = 22,500 simulations) to test species boundaries and reconstruct the evolutionary history of what was expected to be a simple case study. Instead, our results indicate a history of allopatric divergence, secondary contact, introgression, and endemic hybrid speciation driven by the final closure of the Isthmus of Panama and the strengthening of the Gulf Stream Current ~3.5 million years ago. The history of this species complex recovered by model-based methods that allow reticulation differs from that recovered by standard phylogenetic analyses and is unexpected given contemporary distributions. The geologically and biologically meaningful insights gained by our model selection analyses illuminate a novel pathway of species formation that resulted from one of the most biogeographically significant events in Earth’s history.

The mutualism between clownfishes (or anemonefishes) and their giant host sea anemones are among the most immediately recognizable animal interactions on the planet and have attracted a great deal of popular and scientific attention [[1][1]-[5][2]]. However, our evolutionary understanding of this iconic symbiosis comes almost entirely from studies on clownfishes— a charismatic group of 28 described species in the genus Amphiprion [[2][3]]. Adaptation to venomous sea anemones (Anthozoa: Actiniaria) provided clownfishes with novel habitat space, ultimately triggering the adaptive radiation of the group [[2][3]]. Clownfishes diverged from their free-living ancestors 25-30 MYA with their adaptive radiation to sea anemones dating to 13.2 MYA [[2][3], [3][4]]. Far from being mere habitat space, the host sea anemones also receive substantial benefits from hosting clownfishes, making the mutualistic and co-dependent nature of the symbiosis well established [[4][5], [5][2]]. Yet the evolutionary consequences of mutualism with clownfishes have remained a mystery from the host perspective. Here we use bait-capture sequencing to fully resolve the evolutionary relationships among the 10 nominal species of clownfish-hosting sea anemones for the first time ([Figure 1][6]). Using time-calibrated divergence dating analyses we calculate divergence times of less than 25 MYA for each host species, with 9 of 10 host species having divergence times within the last 13 MYA ([Figure 1][6]). The clownfish-hosting sea anemones thus diversified coincidently with clownfishes, potentially facilitating the clownfish adaptive radiation, and providing the first strong evidence for co-evolutionary patterns in this iconic partnership.### Competing Interest StatementThe authors have declared no competing interest. [1]: #ref-1 [2]: #ref-5 [3]: #ref-2 [4]: #ref-3 [5]: #ref-4 [6]: #F1

Ten described species of sea anemones (Anthozoa: Hexacorallia: Actiniaria) serve as hosts to charismatic clownfishes (or anemonefishes) on coral reefs throughout the tropical Indo-West Pacific. Although not diverse in number, the clownfishhosting sea anemones have large biogeographic ranges, exhibit extensive intraspecific phenotypic appearances, and have been surrounded by a great deal of historical and contemporary taxonomic and nomenclatural confusion. We believe these factors have created challenges for field scientists making real-time species-level identifications of host sea anemones. Subsequently, a surprising amount of peer-reviewed clownfish literature never accounts for the host sea anemone, omitting critical data for understanding the symbiosis ecologically and evolutionarily. Here, we leverage the revolution that has taken place in the realm of digital underwater photography over the past 30 years to provide an updated, practical field guide for the clownfish-hosting sea anemones. First however, we review and revise the nomenclature for each species to better reflect valid changes that were made in the historical literature but never broadly adopted. Next, we demonstrate that machine learning algorithms may be of limited use for automating sea anemone species IDs from digital photographs alone-highlighting the importance of organismal expertise for identifying these animals. Finally, we present highresolution digital photographs that encompass much of the intraspecific phenotypic variation encountered underwater, discuss important characteristics useful for field IDs, and provide updated range maps for each species to better reflect the known biogeographic range of each host anemone. We hope the increased confidence in field identification provided by this guide will result in more papers incorporating the sea anemone host data into research frameworks and subsequent publications.

Ecologists have long sought general explanations for the co-occurrence of ecologically similar taxa. Niche theory explains co-occurrence via functional differences among taxa that reduce competition and promote resource partitioning. Alternatively, the unified neutral theory of biodiversity and biogeography suggests that co-occurrence can be attributed to stochastic processes, and thus, presupposes that ecologically similar species that occur in sympatry are functionally analogous. We test these alternative hypotheses in multiple dimensions using the most diverse crustacean-sea anemone symbiosis from coral reefs in the Tropical Western Atlantic. δ 13 C and δ 15 N stable isotope analyses of six crustacean symbionts that co-occur around the host anemone Bartholomea annulata exhibit highly differentiated isotopic niche space spanning three trophic levels. As multiple crustacean species within the symbiosis have been documented as cleaners that remove parasites from reef fishes, we extended our investigation into the broader cleaner community. Our stable isotope analyses of cleaners shows that only Pederson’s cleaner shrimp Ancylomenes pedersoni exhibits δ 15 N isotopic signatures that are consistent with a dedicated cleaning lifestyle. Co-occurring species that have been previously described to clean reef fishes such as Periclimenes yucatanicus, Stenopus hispidus, and Stenorhynchus seticornis all occupy trophic levels well below An. pedersoni . Taken together, our data are consistent with the expectations of niche theory: co-occurring symbiotic crustaceans have highly partitioned niche space with low levels of functional redundancy. Finally, our findings reinforce and extend the ecological importance of An. pedersoni as likely the only dedicated cleaner shrimp on coral reefs in the Tropical Western Atlantic.

Recreational SCUBA diving is widespread and increasing on coral reefs worldwide. Standard open-circuit SCUBA equipment is inherently noisy and, by seeking out areas of high biodiversity, divers inadvertently expose reef communities to an intrusive source of anthropogenic noise. Currently, little is known about SCUBA noise as an acoustic stressor, and there is a general lack of empirical evidence on community-level impacts of anthropogenic noise on coral reefs. Here, we conducted a playback experiment on Caribbean reefs to investigate impacts of SCUBA noise on fish communities and interspecific cooperation at ecologically important cleaning stations of the Pederson’s cleaner shrimp Ancylomenes pedersoni . When exposed to SCUBA-noise playback, the total occurrence of fishes at the cleaning stations decreased by 7%, and the community and cleaning clientele compositions were significantly altered, with 27% and 25% of monitored species being affected, respectively. Compared with ambient-sound playback, SCUBA-noise playback resulted in clients having to wait 29% longer for cleaning initiation and receiving 43% less cleaning; however, cheating, signalling, posing and time spent cleaning were not affected by SCUBA-noise playback. Our study is the first to demonstrate experimentally that SCUBA noise can have at least some negative impacts on reef organisms, confirming it as an ecologically relevant pollutant. Moreover, by establishing acoustic disturbance as a likely mechanism for known impacts of diver presence on reef animals, we also identify a potential avenue for mitigation in these valuable ecosystems.

The marine aquarium trade in the United States operates primarily in Florida, and though the trade’s effects on many marine taxa are largely unknown, local declines have been observed for some harvested species. To reverse local declines in the abundance of the giant Caribbean sea anemone Condylactis gigantea, state managers prohibited harvest of this popular aquarium organism in late 2012. To assess the recovery of C. gigantea following the moratorium, we monitored relative abundance at 45 fixed locations in South Florida from 2013 to 2016. Seventeen of the sites were locations provided to us by commercial aquarium-trade collectors. The remaining sites were locations at which C. gigantea was presumably not being exploited. We found increased densities at only 4 of those 17 sites, while densities at the remaining sites remained unchanged. We also examined genome-wide single nucleotide polymorphism data to assess genetic diversity and population structure of 82 individuals from seven locations (three collection and four non-collection locations) across South Florida. Observed genetic diversity was comparable in all locations. But the location in the eastern Gulf of Mexico showed a significant FIS value, suggesting inbreeding that might be attributed to a small number of occupants. Condylactis gigantea is generally well mixed across South Florida, though a relatively weak pattern of genetic differentiation was detected. These results suggest limited reproductive success and dispersal that is restricted by hydrological and geographical barriers. We highlight the importance of periodic population and genetic monitoring to assess changes in relative abundance and genetic diversity.