Contiguous and complete assemblies of <i>Blastocystis</i> gut microbiome–associated protists reveal evolutionary diversification to host ecology

<i>Blastocystis</i>, 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 <i>Blastocystis</i> 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 <i>Blastocystis.</i> 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 <i>Blastocystis</i>, 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 <i>Blastocystis</i> that has contributed to host-related adaptation. Together, these results highlight substantial genetic and metabolic diversity within the <i>Blastocystis</i> genus, indicating that different lineages of <i>Blastocystis</i> have varied ecological roles in the host gut.