Award: OCE-1924527

Intellectual merit. In larger organisms such as plants and animals, species boundaries can be determined by whether breeding individuals produce fertile offspring (referred to as the biological species concept). Most microbes, especially protozoa and algae, are not amenable to laboratory cultivation and cross-breeding, so we have to infer species identity in wild populations. This is made more difficult by the fact that most of these organisms are very small and hence do not always show discernible species-specific morphological features. In recent years, people have used specific DNA sequences as barcodes to identify species of microbes, but we do not know what degree of difference in these sequences constitutes a clear difference in species identity. This project addressed the species boundary problem using marine planktonic ciliates as a model. These single-celled organisms are key members of the planktonic food web in the ocean, serving as both grazers of phytoplankton and food for higher organisms such as small crustaceans and larval fish. Previous work, also continued in the present project, demonstrated a very high degree of diversity in these organisms based on the number of different barcode sequences observed. In addition to standard barcoding of samples using the ribosomal genes, we used single-cell -omics (genomics and transcriptomics) and metatranscriptomics to quantify ciliate diversity. Single-cell genomics allowed us to obtain a substantial portion of the entire genome for single cells picked either from wild populations or previously isolated cultures. Single-cell transcriptomics allowed us to measure which of the genes present in the genome were actually being expressed, again in both field and laboratory populations. Many ciliates express their genes only after unscrambling them from the genome (portions of the gene may be reversed in the genome or expressed in a different order from what is in the genome). Because the scrambling process would have to be unique to a give species, we can use genome/transcriptome comparisons of single cells to define species boundaries. We showed that two tintinnid ciliate species that look almost identical, Schmidingerella arcuata and S. meunieri, scramble and unscramble their genes differently in the step before transcription, verifying that they represent two distinct biological species. We are also using the single-cell -omics data to better understand the evolutionary relationships (phylogenomics) among tintinnids and related planktonic ciliates. The third method we relied on for studying ciliate diversity is metatranscriptomics. Unlike the single-cell transcriptome, which is unique to a given cell from a given species, the metatranscriptome represents all of the expressed genes from all of the species in a sample of the plankton. To decipher which expressed genes belong to which species, we use phylogenetic methods. These methods compare individual transcriptome sequences pairwise and make trees of relatedness (similarity in sequence) for each gene that can be used to identify where they came from. In other words, all of the versions of a given gene that are from ciliates in the same genus will branch out on the tree together, all those from the same family will branch out together at a higher level, and ultimately all ciliate sequences will branch out together on a large trunk. Our field observations for this project consisted primarily of a year-long time series documenting ciliate diversity weekly from the UConn dock in Groton CT and a cruise across the New England continental shelf in June 2022 aboard the RV Connecticut. We observed subtle differences in the abundances of different species, or variants within species, based on seasons of the year and distance across the shelf. These differences may indicate adaptations to different niches by closely-related ciliate species. Broader Impacts. At the University of Connecticut, this project supported one PhD student, one post-doctoral scholar, one Masters student. We also hosted one undergraduate student from the University of Alaska, who did a summer project on the ciliates that inhabit the respiratory tract of beluga whales. This student, who is a member of the Yupik community of western Alaska, sampled whales at Mystic Aquarium and was a co-author on a journal article that resulted in part from her work. At Hofstra University, a Primarily Undergraduate Institution, the project supported one undergraduate and one master student (both female, of Caribbean descent, and first-generation students) to conduct summer research. The project also supported four undergraduates to present their research at one regional and one on-line international conference, and has provided data for student co-authorship in one published paper and two papers in preparation. In total, the project has generated 9 published articles and several manuscripts in preparation. Last Modified: 11/13/2023 Submitted by: GeorgeBMcmanus