Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts

<i>Bodo</i> is a cosmopolitan genus of free-living bacterivorous single-celled flagellates in the class Kinetoplastea. Genus <i>Bodo</i> is considered the closest free-living lineage to the parasitic lineages <i>Trypanosoma</i> and <i>Leishmania</i>, the causative agents of the human diseases sleeping sickness, Chagas disease and leishmaniasis. Currently, a single genome exists for the one formally described species in the genus, <i>Bodo saltans</i>. Previous studies on <i>B. saltans</i> have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, '<i>Candidatus</i> Bodocaedibacter vickermanii'. Using single-cell sequencing, we isolated, sequenced and assembled genomes for seven uncultured <i>Bodo</i> spp. cells from a freshwater sample from Royal Leamington Spa, UK. Using comparative genomics, we show that these seven cells represent three potentially novel <i>Bodo</i> species exhibiting unexpected levels of diversity at the genome level. Our results indicate that small subunit ribosomal DNA sequencing, often used to classify <i>Bodo</i> flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven <i>Bodo</i> spp. cells. Surprisingly, these seven endosymbionts also represent three novel species, congruent with the phylogeny of the host and exhibiting lineage-specific adaptations. This diversity and host-symbiont association would be indistinguishable in routinely used metabarcoding or bulk sequencing pipelines, thus demonstrating the power of single-cell sequencing to reveal diversity within lineages of microbial eukaryotes.