Abigail L. Lind1,2, Nathan A. McDonald2,3, Elias R. Gerrick4, Ami S. Bhatt5,6 and Katherine S. Pollard1,7,8 1Gladstone Institute for Data Science and Biotechnology, Gladstone Institutes, San Francisco, California 94158, USA; 2School of Biological Sciences, Georgia Institute of Technology, Atlanta, Georgia 30332, USA; 3Department of Biology, Stanford University, Stanford, California 94305, USA; 4Department of Microbiology, University of Chicago, Chicago, Illinois 60637, USA; 5Department of Genetics, Stanford University, Stanford, California 94305, USA; 6Department of Medicine (Hematology, Blood and Marrow Transplantation), Stanford University, Stanford, California 94304, USA; 7Department of Epidemiology and Biostatistics, University of California, San Francisco, California 94158, USA; 8Chan Zuckerberg Biohub SF, San Francisco, California 94158, USA Corresponding author: katherine.pollardgladstone.ucsf.edu Abstract

Blastocystis, an obligate host-associated protist, is the most common microbial eukaryote in the human gut, and is widely distributed across vertebrate hosts. The evolutionary transition of Blastocystis from its free-living stramenopile ancestors to a radiation of host-associated organisms is poorly understood. To explore this, we cultured and sequenced eight strains representing the significant phylogenetic diversity of the genus using long-read, short-read, and Hi-C DNA sequencing, alongside gene annotation and RNA sequencing. Comparative genomic analyses reveal significant variation in gene content and genome structure across Blastocystis. Notably, three strains from herbivorous tortoises, phylogenetically distant from human subtypes, have markedly larger genomes with longer introns and intergenic regions, and retain canonical stop codons absent in the human-associated strains. Despite these genetic differences, all eight isolates exhibit gene losses linked to the reduced cellular complexity of Blastocystis, including losses of cilia and flagella genes, microtubule motor genes, and signal transduction genes. Isolates from herbivorous tortoises contain higher numbers of plant carbohydrate-metabolizing enzymes, suggesting that, like gut bacteria, these protists ferment plant material in the host gut. We find evidence that some of these carbohydrate-metabolizing enzymes were horizontally acquired from bacteria, indicating that horizontal gene transfer is an ongoing process in Blastocystis that has contributed to host-related adaptation. Together, these results highlight substantial genetic and metabolic diversity within the Blastocystis genus, indicating that different lineages of Blastocystis have varied ecological roles in the host gut.

Footnotes

[Supplemental material is available for this article.]

Article published online before print. Article, supplemental material, and publication date are at https://www.genome.org/cgi/doi/10.1101/gr.279080.124.

Freely available online through the Genome Research Open Access option.

Received February 7, 2024. Accepted April 10, 2025.