My research is focused on the Systematics of Symbioses. I use modern genomic approaches (i.e. reduced representation sequencing, bait-capture probes, & amplicon sequencing) to understand the evolution of tropical marine symbioses at the species, population, genomic and behavioral levels. My research program can be broadly categorized in to five main themes: 1) phylogenetics and species delimitation, 2) comparative phylogeography & population genetics 3) developing and testing new genomic approaches to study the evolution of symbiotic anthozoans, 4) microbial ecology of the clownfish-sea anemone symbiosis, and 5) the evolution and ecology of mutualistic behaviors.
Organismally, my research program is centered on iconic sea anemone symbioses from tropical coral reefs. In the Indian and Pacific Oceans, I study the evolution of the clownfish-hosting sea anemones- a group that has been largely overlooked, although they form model mutualisms with ~30 species of clownfishes and are highly sought after in the ornamental aquarium trade. In the Caribbean and Tropical Western Atlantic I study the evolution of symbioses between sea anemones and their crustacean symbionts- some of which are ecologically important cleaner species that remove parasites from >20 families of common reef fishes.
1. Phylogenetics and Species Delimitation
Symbioses with fully disentangled systematics provide powerful research frameworks for evaluating fundamental biological processes, yet until now, no major, modern (post-1980’s) effort has focused on the systematics and evolution of tropical sea anemones and their non-fish symbionts. Sea anemone symbioses are diverse, among the most recognizable organisms on the planet, perform important ecosystem services, and contribute meaningfully to tropical marine biodiversity. As for many poorly studied invertebrates, it is expected these lineages of anemones and their symbionts are more species rich than is currently recognized. As sea anemones and their symbionts have been used as models for understanding core concepts across scientific disciplines, the discovery of cryptic taxa can have wide-reaching implications.
a) Tropical Sea Anemone Biodiversity- Contrary to the generally observed pattern of hyperdiversity in the tropics, anemone diversity peaks in temperate ecosystems. As a result, tropical species have received a fraction of the scientific attention as temperate ones, with no major efforts in the systematics of tropical anemone diversity since the advent of modern DNA sequencing. Compounding a lack of systematic effort in the tropics, anemones have few diagnostic morphological characters and slowly evolving mitochondrial DNA that is ineffective as a species-level marker. Thus, it is likely that tropical anemone diversity is vastly under-described, and a genomic approach is required to simultaneously delimit species, place them into broader phylogenomic context, and determine what morphological characters are informative at every hierarchical level. To date, my genomic research has shown that a common and ecologically important sea anemone in the Caribbean is a species complex (Titus et al. In Press; Molecular Ecology), and in the largest phylogenetic study of sea anemones, I’ve shown that the symbiosis with clownfishes has evolved independently at least three times in sea anemones, and that some host anemone lineages have different biogeographic origins than the clownfises themselves (Titus et al., 2019; Mol. Phylogenet. Evol.). My ongoing research at AMNH into tropical anemone diversity is revealing that many of the nominal clownfish-hosting anemones are in fact cryptic species complexes. I also continue to collaborate on Caribbean sea anemone diversity projects with the Florida Fish and Wildlife Conservation Commission.
b) Cryptic speciation in a crustacean-sea anemone symbioses– In the Caribbean, sea anemones host a diverse community of crustacean symbionts. Some of which are ecologically important cleaner shrimp that remove parasites from reef fishes. Although these are highly recognizable and have been used in dozens of studies of cleaning symbioses, they have been poorly studied genetically. My research has highlighted extensive cryptic species diversity in a common, yet understudied, sea anemone symbiosis in the Caribbean. In the Caribbean, my work has more than doubled the recognized extant diversity in the corkscrew sea anemone (Bartholomea annulata) symbiosis. I have delimited nine new lineages in four nominal crustacean and anemone species (e.g. Titus & Daly, 2015 Mar. Ecol., 2017a Coral Reefs, b Reef Encounter; Titus et al., 2017 Biol. J. Linn. Soc. ; Titus et al., 2018a J. Biogeo., b Mol. Ecol. ). Interestingly, although my research demonstrates the importance of symbiosis evolutionarily, among specialist anemone symbionts, other processes (i.e. allopatry & ecological speciation) are responsible for the extant species-level diversity in these complexes.
2. Comparative Phylogeography and Population Genetics
Highly specialized symbiotic interactions are thought to reflect the cumulative effect of co-evolutionary processes (e.g. shared history and natural selection). At deep evolutionary levels (≥ species), co-evolutionary studies inform us of the spatiotemporal outcomes of symbioses. Comparative population-level studies hold the potential to inform us of the processes that generate and maintain these interactions, and the role of symbiosis at the early stages of divergence. Symbioses generate a priori hypotheses that tightly linked ecological communities co-evolve spatially and temporally. To test this hypothesis in the TWA I used anemone host B. annulata and five crustacean symbionts that vary in their symbiotic association to it. Comparative phylogeographic patterns downplay the role of symbiotic specialization in generating intraspecific patterns of diversity across the range (Titus & Daly 2017, Coral Reefs; Titus et al., In Prep). On a fine scale, sequence data reflects idiosyncratic expansion events along the Florida Reef Tract (Titus & Daly 2017 Coral Reefs). Using NSF funding to expand sampling and sequencing efforts, range-wide ddRADseq data reflect some shared phylogeographic barriers, but these are not all shared with the anemone host. Coalescent model selection reflects discordant demographic processes leading to diversification at different temporal scales (Titus et al. In Prep).
3. Anthozoan Population Genomics
Although the field of population genetics and phylogeography has a long history in marine systems, cnidarians in the class Anthozoa have been historically challenging to work with at the population level. In addition to large range sizes and the logistical difficulties of sampling underwater, mitochondrial DNA barcodes (mtDNA), the molecular marker of choice for metazoan phylogeographic studies from the field’s outset, evolve too slowly in most anthozoans to be useful for intraspecific studies. The development of high-throughput sequencing and the generation of thousands of single nucleotide polymorphisms via reduced representation approaches (i.e. RADseq) has revolutionized population genomics for non-model species. However, most tropical anthozoans found on coral reefs form endosymbioses with photosynthetic dinoflagellates in the family Symbiodinaceae. In field-collected samples, contamination from symbiodiniaceans is unavoidable, and resulting DNA extractions harbor a mix of anthozoan and dinoflagellete DNA (termed “holobiont DNA”). The combination of slowly evolving mtDNA and dinoflagellate contamination complicates the development of molecular markers suitable for population level questions unless a reference genome is available to parse anthozoan from algal sequences. Recently, I have made a key insight into using reduced representation sequencing approaches for symbiotic anthozoans which indicates that under many common circumstances, a reference genome is not necessary to parse anthozoan form algal DNA to make robust phylogeographic inferences using RADseq (Titus & Daly, 2018, biorxiv). In short, when the focal anthozoan species harbors multiple genera of Symbiodineaceans, under moderately conserved missing data thresholds, de novo SNP calling pipelines such as ipyRAD & Stacks cannot find enough conserved, homologous, symbiodineacean loci to be retained in the final SNP dataset. The vast majority of the SNPs are thus target anthozoan sequences and any remaining symbiont DNA is simply genetic noise. I am continuing this vein of research by testing the utility of recently developed bait-capture proves targeting ultra-conserved elements for anthozoans for population-level studies (Quattrini et al., In Prep).
4. Microbial Ecology of the Clownfish-Sea Anemone Symbiosis
All multicellular life engages in symbioses with prokaryotic microbiota that are essential for survival, critically impact individual health, development, and nutrient acquisition, and which serve as a primary interface between individuals and their environment. The composition and diversity of an individual’s microbial community (i.e. the microbiome) has been shown to be influenced by a variety of factors, but are generally discussed as a combination of evolutionary history and ecology. Using the iconic clownfish symbiosis, I am conducting the first comparative study of the clownfish-hosting sea anemone microbiome. Using 16s amplicon sequencing of five species of co-occurring sea anemones in the Maldives, I am testing the importance of host identity, clownfish symbiont association, and habitat on the genetic and predicted functional diversity of the microbiome. Our data indicate that anemone host identity structures the genetic diversity of the microbiome, but the presence of clownfishes increases the predicted functional diversity of the host over anemones that do not host fish. These data are the first evidence that suggests a previously unknown benefit provided to host anemones from clownfish symbionts (Titus et al., In Prep). I have continued the microbial research of the clownfish-hosting sea anemones in collaboration with scientists from CRIOBE (Moorea, French Polynesia) and the Smithsonian Institution to explore the genomic basis of bleaching resistance in the clownfish hosting sea anemones in Moorea.
5. Ecology and Evolution of Mutualistic Cleaning Behaviors
Sea anemone symbioses are not only useful systems for understanding the genomics of symbiosis on coral reefs, but are also model systems for understanding the evolution and ecology of cooperative behaviors. To that end, I have developed a stream of research in collaboration with Dr. Dan Exton and biodiversity NGO Operation Wallacea (UK) on cleaner shrimp behavioral ecology and evolution. Using remotely deployed underwater video cameras, we have explored various aspects of the cleaning symbiosis in the Caribbean including the effects of diver presence (Titus et al., 2015a, PLOS One), temporal patterns of cleaning activity (Titus et al., 2015b, Mar. Biol. ), possibly mimicry within cleaner shrimps (Titus et al., 2017c, Roy. Soc. Open Sci.), and the evolution of cheating behaviors across ocean basins (Titus et al, 2019a, Sci. Rep.). Ongoing work at our field site in Utila, Honduras has been ongoing from 2013-present.