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

Bodo is a cosmopolitan genus of free living bacterivorous single-celled flagellates in the class Kinetoplastea. Members of genus Bodo are considered the closest free-living relatives to the parasitic lineages Trypanosoma and Leishmania, 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, Bodo saltans. Previous studies on B. saltans have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, "Candidatus Bodocaedibacter vickermanii". Using single cell-sequencing, we isolated, sequenced, and assembled genomes for seven uncultured Bodo spp. cells from a single freshwater sample from Royal Leamington Spa, UK. By using comparative genomics, we show that these seven cells represent three potentially novel Bodo species and exhibit unexpected levels of diversity at the genome level. Our results indicate that Small Subunit (SSU) rDNA sequencing, often used to classify Bodo flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven Bodo spp. cells. Surprisingly, these seven bacterial endosymbionts also represent three potentially novel species and one novel genus of Holosporales bacteria. This diversity would be indistinguishable in routinely-used SSU ribosomal DNA (rDNA) metabarcoding or bulk sequencing pipelines, thus demonstrating the utility of using single-cell sequencing to reveal the level of genomic diversity within lineages of microbial eukaryotes and their cobionts.