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

## TL;DR

Single-cell sequencing uncovered new genetic diversity in Bodo flagellates and their bacterial symbionts, showing that traditional methods miss this complexity.

## Contribution

The study reveals three potential new Bodo species and their associated novel bacterial endosymbionts using single-cell sequencing.

## Key findings

- Seven Bodo spp. cells represent three potentially novel species with high genetic diversity.
- All seven Bodo cells contained a Holosporales bacterium, representing three novel endosymbiont species.
- Traditional classification methods like small subunit ribosomal DNA sequencing are insufficient for Bodo species delimitation.

## Abstract

Bodo is a cosmopolitan genus of free-living bacterivorous single-celled flagellates in the class Kinetoplastea. Genus Bodo is considered the closest free-living lineage 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 freshwater sample from Royal Leamington Spa, UK. Using comparative genomics, we show that these seven cells represent three potentially novel Bodo species exhibiting unexpected levels of diversity at the genome level. Our results indicate that small subunit ribosomal DNA 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 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.