Population genomics for symbiotic anthozoans: can reduced representation approaches be used for taxa without reference genomes?

Population genetic studies of symbiotic anthozoans have been historically challenging because their endosymbioses with dinoflagellates have impeded marker development. Genomic approaches like reduced representation sequencing alleviate marker development issues but produce anonymous loci, and without a reference genome, it is unknown which organism is contributing to the observed patterns. Alternative methods such as bait-capture sequencing targeting Ultra-Conserved Elements are now possible but costly. Thus, RADseq remains attractive, but how useful are these methods for symbiotic anthozoan taxa without a reference genome to separate anthozoan from algal sequences? We explore this through a case-study using a double-digest RADseq dataset for the sea anemone Bartholomea annulata. We assembled a holobiont dataset (3854 loci) for 101 individuals, then used a reference genome to create an aposymbiotic dataset (1402 loci). For both datasets, we investigated population structure and used coalescent simulations to estimate demography and population parameters. We demonstrate complete overlap in the spatial patterns of genetic diversity, demographic histories, and population parameter estimates for holobiont and aposymbiotic datasets. We hypothesize that the unique combination of anthozoan biology, diversity of the endosymbionts, and the manner in which assembly programs identify orthologous loci alleviates the need for reference genomes in some circumstances. We explore this hypothesis by assembling an additional 21 datasets using the assembly programs pyRAD and Stacks. We conclude that RADseq methods are more tractable for symbiotic anthozoans without reference genomes than previously realized.

Novel in situ observations of asexual reproduction in the carpet sea anemone, Stichodactylamertensii (Stichodactylidae, Actiniaria).

Merten's carpet sea anemone, Stichodactylamertensii Brandt, 1835, is the largest known sea anemone species in the world, regularly exceeding one meter in oral disc diameter. A tropical species from the Indo-Pacific, S.mertensii drapes prominently over coral reef substrates and is a common host to numerous species of clownfishes and other symbionts throughout its range, which extends from the Red Sea through the Central Pacific Ocean. Long thought to reproduce via sexual reproduction only, recent genetic evidence suggests it may rarely reproduce asexually as well, although this process had never been confirmed through direct observation and the mechanism was yet to be described. Here, we directly observed and documented in situ asexual fragmentation via budding, in real time, by a Red Sea S.mertensii in a turbid inshore reef environment. While asexual reproduction is not unusual in sea anemones as a group, it is typically expected to be uncommon for large-bodied species. Herein, we describe S.mertensii fragmentation, provide high resolution images of the event from the Saudi Arabian coastline at multiple time points, and confirm asexual reproduction for this species.

Comparison of sequence-capture and ddRAD approaches in resolving species and populations in hexacorallian anthozoans.

Genome-level sequencing is the next step in understanding species-level relationships within Anthozoa (soft corals, anemones, stony corals, and their kin) as morphological and PCR-directed (single-locus) sequencing methods often fall short of differentiating species. The sea anemone genus Metridium is a common northern temperate sea anemone whose species are difficult to differentiate using morphology alone. Here we use Metridium as a case study to confirm the low level of information available in six loci for species differentiation commonly sequenced for Actiniaria and explore and compare the efficacy of ddRAD and sequence-capture methods in species-level systematics and biogeographic studies. We produce phylogenetic trees from concatenated datasets and perform DAPC and STRUCTURE analyses using SNP data. The six conventional loci are not able to consistently differentiate species within Metridium. The sequence-capture dataset resulted in high support and resolution for both current species and relationships between geographic areas. The ddRAD datasets displayed ambiguity among species, and support between major geographic groupings was not as high as the sequence-capture datasets. The level of resolution and support resulting from the sequence-capture data, combined with the ability to add additional individuals and expand beyond the genus Metridium over time, emphasizes the utility of sequence-capture methods for both systematics and future biogeographic studies within anthozoans. We discuss the strengths and weaknesses of the genomic approaches in light of our findings and suggest potential implications for the biogeography of Metridium based on our sampling.

Multiple cleaner species provide simultaneous services to coral reef fish clients.

Cleaning symbioses on tropical coral reefs are typically documented between two species: a single client fish and one or more conspecific cleaners. However, multiple cleaner species living sympatrically in the Caribbean have been anecdotally reported to simultaneously clean the same client. Nothing is known about the patterns and processes driving these interactions, which may differ from those involving a single cleaner species. Here, we used remote underwater videography on three reefs in Honduras to record simultaneous cleaning interactions involving Pederson's cleaner shrimp (Ancylomenes pedersoni) and cleaner gobies (Elacatinus spp.). A pilot study on adjacent shrimp and goby stations found interactions were always initiated by shrimp. A larger, multi-year dataset shows cleaner gobies joined 28% of all interactions initiated at A. pedersoni cleaning stations with cleaner gobies residing nearby. Client body size significantly predicted simultaneous cleaning interactions, with 45% of interactions simultaneous for clients greater than 20 cm total body length compared with only 8% for clients less than 20 cm. We also found that simultaneous cleaning interactions lasted over twice as long as shrimp-only interactions. We propose these novel multi-species interactions to be an ideal model system to explore broader questions about coexistence, niche overlap and functional redundancy among sympatric cleaner species.

Building a global genomics observatory: Using GEOME (the Genomic Observatories Metadatabase) to expedite and improve deposition and retrieval of genetic data and metadata for biodiversity research.

Genetic data represent a relatively new frontier for our understanding of global biodiversity. Ideally, such data should include both organismal DNA-based genotypes and the ecological context where the organisms were sampled. Yet most tools and standards for data deposition focus exclusively either on genetic or ecological attributes. The Genomic Observatories Metadatabase (GEOME: geome-db.org) provides an intuitive solution for maintaining links between genetic data sets stored by the International Nucleotide Sequence Database Collaboration (INSDC) and their associated ecological metadata. GEOME facilitates the deposition of raw genetic data to INSDCs sequence read archive (SRA) while maintaining persistent links to standards-compliant ecological metadata held in the GEOME database. This approach facilitates findable, accessible, interoperable and reusable data archival practices. Moreover, GEOME enables data management solutions for large collaborative groups and expedites batch retrieval of genetic data from the SRA. The article that follows describes how GEOME can enable genuinely open data workflows for researchers in the field of molecular ecology.

Host identity and symbiotic association affects the taxonomic and functional diversity of the clownfish-hosting sea anemone microbiome.

All eukaryotic life engages in symbioses with a diverse community of bacteria that are essential for performing basic life functions. In many cases, eukaryotic organisms form additional symbioses with other macroscopic eukaryotes. The tightly linked physical interactions that characterize many macroscopic symbioses create opportunities for microbial transfer, which likely affects the diversity and function of individual microbiomes, and may ultimately lead to microbiome convergence between distantly related taxa. Here, we sequence the microbiomes of five species of clownfish-hosting sea anemones that co-occur on coral reefs in the Maldives. We test the importance of evolutionary history, clownfish symbiont association, and habitat on the taxonomic and predicted functional diversity of the microbiome, and explore signals of microbiome convergence in anemone taxa that have evolved symbioses with clownfishes independently. Our data indicate that host identity and clownfish association shapes the majority of the taxonomic diversity of the clownfish-hosting sea anemone microbiome, and predicted functional microbial diversity analyses demonstrate a convergence among host anemone microbiomes, which reflect increased functional diversity over individuals that do not host clownfishes. Further, we identify upregulated predicted microbial functions that are likely affected by clownfish presence. Taken together our study potentially reveals an even deeper metabolic coupling between clownfishes and their host anemones, and what could be a previously unknown mutualistic benefit to anemones that are symbiotic with clownfishes.

Genomic signatures of sympatric speciation with historical and contemporary gene flow in a tropical anthozoan (Hexacorallia: Actiniaria).

Sympatric diversification is recognized to have played an important role in the evolution of biodiversity. However, an in situ sympatric origin for codistributed taxa is difficult to demonstrate because different evolutionary processes can lead to similar biogeographic outcomes, especially in ecosystems that can readily facilitate secondary contact due to a lack of hard barriers to dispersal. Here we use a genomic (ddRADseq), model-based approach to delimit a species complex of tropical sea anemones that are codistributed on coral reefs throughout the Tropical Western Atlantic. We use coalescent simulations in fastsimcoal2 and ordinary differential equations in Moments to test competing diversification scenarios that span the allopatric-sympatric continuum. Our results suggest that the corkscrew sea anemone Bartholomea annulata is a cryptic species complex whose members are codistributed throughout their range. Simulation and model selection analyses from both approaches suggest these lineages experienced historical and contemporary gene flow, supporting a sympatric origin, but an alternative secondary contact model receives appreciable model support in fastsimcoal2. Leveraging the genome of the closely related Exaiptasia diaphana, we identify five loci under divergent selection between cryptic B. annulata lineages that fall within mRNA transcripts or CDS regions. Our study provides a rare empirical, genomic example of sympatric speciation in a tropical anthozoan and the first range-wide molecular study of a tropical sea anemone, underscoring that anemone diversity is under-described in the tropics, and highlighting the need for additional systematic studies into these ecologically and economically important species.

Phylogenetic relationships among the clownfish-hosting sea anemones.

The clownfish-sea anemone symbiosis has been a model system for understanding fundamental evolutionary and ecological processes. However, our evolutionary understanding of this symbiosis comes entirely from studies of clownfishes. A holistic understanding of a model mutualism requires systematic, biogeographic, and phylogenetic insight into both partners. Here, we conduct the largest phylogenetic analysis of sea anemones (Order Actiniaria) to date, with a focus on expanding the biogeographic and taxonomic sampling of the 10 nominal clownfish-hosting species. Using a combination of mtDNA and nuDNA loci we test (1) the monophyly of each clownfish-hosting family and genus, (2) the current anemone taxonomy that suggests symbioses with clownfishes evolved multiple times within Actiniaria, and (3) whether, like the clownfishes, there is evidence that host anemones have a Coral Triangle biogeographic origin. Our phylogenetic reconstruction demonstrates widespread poly- and para-phyly at the family and genus level, particularly within the family Stichodactylidae and genus Stichodactyla, and suggests that symbioses with clownfishes evolved minimally three times within sea anemones. We further recover evidence for a Tethyan biogeographic origin for some clades. Our data provide the first evidence that clownfish and some sea anemone hosts have different biogeographic origins, and that there may be cryptic species of host anemones. Finally, our findings reflect the need for a major taxonomic revision of the clownfish-hosting sea anemones.

Lack of strategic service provisioning by Pederson's cleaner shrimp (Ancylomenes pedersoni) highlights independent evolution of cleaning behaviors between ocean basins.

Marine cleaning interactions have been useful model systems for exploring evolutionary game theory and explaining the stability of mutualism. In the Indo-Pacific, cleaner organisms will occasionally "cheat" and remove live tissue, clients use partner control mechanisms to maintain cleaner honesty, and cleaners strategically increase service quality for predatory clients that can "punish" more severely. The extent to which reef communities in the Caribbean have evolved similar strategies for maintaining the stability of these symbioses is less clear. Here we study the strategic service provisioning in Pederson's cleaner shrimp (Ancylomenes pedersoni) on Caribbean coral reefs. In the Gulf of Honduras, we use video observations to analyze >1000 cleaning interactions and record >850 incidents of cheating. We demonstrate that A. pedersoni cheat frequently and do not vary their service quality based on client trophic position or cleaner shrimp group size. As a direct analog to the cleaner shrimp A. longicarpus in the Indo-Pacific, our study highlights that although cleaning interactions in both ocean basins are ecologically analogous and result in parasite removal, the strategic behaviors that mediate these interactions have evolved independently in cleaner shrimps.

Comparative behavioural observations demonstrate the 'cleaner' shrimp Periclimenes yucatanicus engages in true symbiotic cleaning interactions.

Cleaner shrimps are ecologically important members of coral reef communities, but for many species, cleaner status (i.e. dedicated, facultative and mimic), clientele and ecological role remain unverified or described. On Caribbean coral reefs, the spotted 'cleaner' shrimp Periclimenes yucatanicus forms symbioses with sea anemones that may serve as cleaning stations for reef fishes. The status of this species as a cleaner is ambiguous: only a single in situ cleaning interaction has been reported, and in the only test of its efficacy as a cleaner, it did not effectively reduce parasite loads from surgeonfish. It has subsequently been hypothesized by other authors to be a cleaner mimic. We conduct a comparative investigation of cleaning behaviour between P. yucatanicus and the ecologically similar, closely related, dedicated cleaner shrimp Ancylomenes pedersoni in Curacao, Netherlands Antilles. We provide the first detailed field observations on cleaning behaviour for P. yucatanicus and test multiple behavioural expectations surrounding mimicry in cleaning symbioses. We found that P. yucatanicus regularly signals its availability to clean, client fishes visit regularly and the shrimp does engage in true symbiotic cleaning interactions, but these are brief and our video reflects a species that appears hesitant to engage posing clients. In comparison to A. pedersoni, P. yucatanicus stations had significantly fewer total visits and cleans, and 50% of all cleaning interactions at P. yucatanicus stations were shorter than 10 s in total duration. Our behavioural observations confirm that P. yucatanicus is a true cleaner shrimp; we reject the hypothesis of mimicry. However, investigation is needed to confirm whether this species is a dedicated or facultative cleaner. We hypothesize that P. yucatanicus has a specialized ecological role as a cleaner species, compared to A. pedersoni.

Do reef fish habituate to diver presence? Evidence from two reef sites with contrasting historical levels of SCUBA intensity in the Bay Islands, Honduras.

Contact between humans and the marine environment is increasing, but the capacity of communities to adapt to human presence remains largely unknown. The popularization of SCUBA diving has added a new dimension to human impacts in aquatic systems and, although individual-level impacts have been identified, cumulative effects on ecosystem function and community-wide responses are unclear. In principle, habituation may mitigate the consequences of human presence on the biology of an individual and allow the quick resumption of its ecological roles, but this has not been documented in aquatic systems. Here, we investigate the short-term impact of human presence and the long-term habituation potential of reef-fish communities to recreational SCUBA divers by studying symbiotic cleaning interactions on coral reefs with differing levels of historical contact with divers. We show that incidences of human contact result in a smaller decline in ecosystem function and more rapid resumption of baseline services on a reef in Utila, Honduras that has heavy historical levels of SCUBA diver presence, compared to an un-dived reef site in the Cayos Cochinos Marine Protected Area (CCMPA). Nonetheless, despite the generally smaller change in ecosystem function and decades of regular contact with divers, cleaning behavior is suppressed by >50% at Utila when divers are present. We hypothesize that community-wide habituation of reef fish is not fully achievable and may be biologically restricted to only partial habituation. Differential responses to human presence impacts the interpretation and execution of behavioral research where SCUBA is the predominant means of data collection, and provides an important rationale for future research investigating the interplay between human presence, ecosystem function, and community structure on coral reefs.