Date of Award

2025

Degree Type

Dissertation

Degree Name

Doctor of Philosophy in Oceanography

Specialization

Biological Oceanography

Department

Oceanography

First Advisor

Roxanne Beinart

Abstract

Ciliates are a diverse group of microbial eukaryotes known for their ability to establish various symbiotic partnerships as well as thrive in a wide array of environments, including oxygen-depleted habitats which range from coastal marine sediment and the digestive tracts of animals to deep-sea hydrothermal vents. Anaerobic ciliates harbor intracellular methanogenic archaea that increase the metabolic efficiency of the host by utilizing host by-products in their own metabolism, which in turn produces methane, a potent greenhouse gas. The acquisition of these symbionts is thought to have facilitated the multiple independent transitions that ciliates have undergone from aerobic to anaerobic lifestyles during their evolution. These symbiotic consortia of ciliates and methanogens are ubiquitous and likely have a large ecological impact via grazing and methane production. Yet, while anaerobic archaea and bacteria are well-studied and appreciated for their roles in global biogeochemical processes, ciliates and other protists are largely unaccounted for in our assessments of these global, growing anoxic microbial communities. There are many fundamental unknowns surrounding these associations, namely an understanding of the full diversity and distribution of anaerobic ciliates as well as the evolutionary dynamics underlying the symbiosis, particularly in marine environments. As the endosymbiotic methanogens of anaerobic ciliates and other protists are the only known intracellular archaea, they represent an excellent opportunity to better understand the evolutionary and functional dynamics of endosymbiosis, most of our current under-standing of which originates from terrestrial, multicellular hosts. This dissertation com-bines global-scale metabarcoding meta-analysis, culture-based symbiont surveys, and comparative genomics to explore the diversity, specificity, and evolutionary history of anaerobic ciliates and their methanogenic symbionts across global anoxic habitats.

Anaerobic ciliates are ubiquitous and diverse and have been detected across a huge array of anoxic marine habitats, including intertidal sediments, cold seeps, and deep-sea basins. However, studies which have identified them are highly localized and virtually nothing is known about their global distribution and patterns of diversity across habitat types. Additionally, most investigations into the diversity of anaerobic ciliates have focused on endobiotic and freshwater species, with much left to be discovered about the diversity of marine lineages. In Chapter 2, I leveraged publicly available 18S rDNA datasets to interrogate the global diversity and distribution of anaerobic ciliates as well as identify and explore potential novel lineages. Using my own custom and reproducible Snakemake pipeline, I reprocessed raw data from ~28000 samples from 43 studies across 19 habitat types to extract ciliate sequences, which we phylogenetically placed onto curated reference phylogenetic trees. I recovered nearly 3200 amplicon sequence variants (ASVs) assigned to anaerobic ciliate lineages across 649 samples. My results highlight the global presence of anaerobic ciliates across geographically and physically disparate marine habitats and suggest that these communities are not structured by depth but more likely influenced by local physicochemical conditions (e.g., redox structure). Additionally, my analyses revealed substantial previously unknown diversity in these communities, particularly within the plagiopylean family Epalxellidae. Furthermore, this study provides a framework for other researchers to investigate the diversity and ecology of other little-known protist lineages.

Most anaerobic ciliates studied thus far have formed partnerships with methanogenic archaea which are vital for their survival in anoxia. However, our knowledge of these symbioses is limited to geographically isolated studies that describe a few known symbionts associated with a few ciliate species, and therefore patterns of symbiont specificity and fidelity are not well known. In Chapters 3 and 4, I sought to deeply survey these host-symbiont partnerships via comprehensive sampling, culturing, and identification and phylogenetic analysis of host-symbiont pairs. Chapter 3 surveyed the symbionts of two commonly co-occurring and divergent anaerobic genera, Metopus and Plagiopyla. By focusing on shallow, intertidal sediments in Southern New England, I minimized geographical and environmental differences and assessed the impact of host phylogeny on symbiont identity. I found that both groups of marine ciliates harbor closely related Methanocorpusculum which are stable at the host species level, but that there was also evidence of symbiont replacements at higher taxonomic levels.

In Chapter 4, I surveyed symbionts across the order Metopida from both fresh-water and marine/brackish habitats in order to evaluate the influence of habitat type and host phylogeny on symbiont identity, both within and among host lineages. My results indicated that each host strain harbored a single methanogen strain and that habitat greatly influenced symbiont identity, with virtually all marine species harboring Methanocorpusculum and freshwater species hosting Methanoregula or Methanobacterium. Additionally, symbionts were specific to host species at the genus level. Taken together, Chapters 3 and 4 greatly expand our knowledge of these symbioses and support a mixed-transmission mode with periodic symbiont swaps/replacements for this common yet complex partnership.

In Chapter 5, I expanded on the results of Chapter 3 and conduct a phylo- and pangenomic analysis of Methanocorpusculum symbionts in order to better understand the evolutionary relationships and functional differences between symbionts from these two divergent anaerobic ciliate lineages. Methanocorpusculum also includes strains which reside in the digestive tracts of a large diversity of animal species as well as strains isolated from the environment. It is one of the few prokaryotic genera that forms mutualistic associations with both metazoans and protists, allowing us to investigate how symbionts evolve under these drastically different selective regimes. I found that ciliate-associated strains were virtually functionally and phylogenetically indistinguishable and retain broad biosynthetic functions. Animal-associated strains formed their own distinct clade and appear to be functionally adapted to the gut environment. My results indicated that ciliate-associated Methanocorpsculum are very similar to their free-living counterparts, potentially due to recent acquisition and/or the impact of mixed-mode transmission on the maintenance of their stable, gene-rich symbiosis, and challenge the notion that intracellular symbiosis necessarily leads to genome erosion.

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