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Volume 12, Issue 3

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

This article is part of the Microbial Genomics of Eukaryotes collection

Abstract

is a cosmopolitan genus of free-living bacterivorous single-celled flagellates in the class Kinetoplastea. Genus is considered the closest free-living lineage to the parasitic lineages and , 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, . Previous studies on have shown that it is dependent on an endosymbiotic bacterium from the order Holosporales, ‘ Bodocaedibacter vickermanii’. Using single-cell sequencing, we isolated, sequenced and assembled genomes for seven uncultured spp. cells from a freshwater sample from Royal Leamington Spa, UK. Using comparative genomics, we show that these seven cells represent three potentially novel species exhibiting unexpected levels of diversity at the genome level. Our results indicate that small subunit ribosomal DNA sequencing, often used to classify flagellates, is insufficient for determining species delimitation in this genus. In addition, we recovered a Holosporales bacterium genome from all seven 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.

  • Received:
  • Accepted:
  • Published Online:
Keyword(s): Bodo saltans , environmental sequencing , holosporales , Kinetoplastea , protist and single-cell genome
Funding
This study was supported by the:
  • Wellcome Trust (Award 218328)
    • Principal Award Recipient: NeilHall
  • Wellcome Trust (Award 226458)
    • Principal Award Recipient: NeilHall
  • Biotechnology and Biological Sciences Research Council (Award BB/CCG1720/1)
    • Principal Award Recipient: NeilHall
  • Biotechnology and Biological Sciences Research Council (Award BB/CCG2220/1)
    • Principal Award Recipient: NeilHall
  • Biotechnology and Biological Sciences Research Council (Award BBX011089/1)
    • Principal Award Recipient: NotApplicable
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/ER/230002B)
    • Principal Award Recipient: NotApplicable
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/T/000PR9816)
    • Principal Award Recipient: KarimGharbi
  • Biotechnology and Biological Sciences Research Council (Award BBS/E/ER/23NB0006)
    • Principal Award Recipient: KarimGharbi
  • Royal Society (Award URF/R/191005)
    • Principal Award Recipient: ThomasA. Richards
© 2026 The Authors
  • This is an open-access article distributed under the terms of the Creative Commons Attribution License. This article was made open access via a Publish and Read agreement between the Microbiology Society and the corresponding author’s institution.
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2026-03-18
2026-04-18

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References

  1. Warring SD, McGowan J, Kilias ES, Lipscombe J, Alacid E et al.Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts Figshare 2026 [View Article]
     [Google Scholar]
  2. Adl SM, Simpson AGB, Lane CE, Lukeš J, Bass D et al. The revised classification of eukaryotes. J Eukaryot Microbiol 2012; 59:429–493 [View Article] [PubMed]
     [Google Scholar]
  3. Moreira D, López-García P, Vickerman K. An updated view of kinetoplastid phylogeny using environmental sequences and a closer outgroup: proposal for a new classification of the class Kinetoplastea. Int J Syst Evol Microbiol 2004; 54:1861–1875 [View Article] [PubMed]
     [Google Scholar]
  4. Simpson AGB, Stevens JR, Lukeš J. The evolution and diversity of kinetoplastid flagellates. Trends in Parasitology 2006; 22:168–174 [View Article]
     [Google Scholar]
  5. Simpson AGB, Lukes J, Roger AJ. The evolutionary history of kinetoplastids and their kinetoplasts. Mol Biol Evol 2002; 19:2071–2083 [View Article] [PubMed]
     [Google Scholar]
  6. Callahan HA, Litaker RW, Noga EJ. Molecular taxonomy of the suborder bodonina (Order Kinetoplastida), including the important fish parasite, Ichthyobodo necator. J Eukaryot Microbiol 2002; 49:119–128 [View Article] [PubMed]
     [Google Scholar]
  7. Deschamps P, Lara E, Marande W, López-García P, Ekelund F et al. Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids. Mol Biol Evol 2011; 28:53–58 [View Article] [PubMed]
     [Google Scholar]
  8. Brooker BE. Fine structure of Bodo saltans and Bodo caiidatus (Zoomastigophora:Protozoa) and their affinities with the Trypanosomatidae. Bull Br Mus Nat Hist Zool 1971; 22:87–102 [View Article]
     [Google Scholar]
  9. Vickerman K, Preston T. Comparative cell biology of the kinetoplastid flagellates. Biol Kinetoplastida 1979; 2:35–130
     [Google Scholar]
  10. Dolezel D, Jirků M, Maslov DA, Lukes J. Phylogeny of the bodonid flagellates (kinetoplastida) based on small-subunit rRNA gene sequences. Int J Syst Evol Microbiol 2000; 50 Pt 5:1943–1951 [View Article] [PubMed]
     [Google Scholar]
  11. von der Heyden S, Chao EE, Vickerman K, Cavalier-Smith T. Ribosomal RNA phylogeny of bodonid and diplonemid flagellates and the evolution of euglenozoa. J Eukaryot Microbiol 2004; 51:402–416 [View Article] [PubMed]
     [Google Scholar]
  12. Blom D, de Haan A, van den Berg M, Sloof P, Jirku M et al. RNA editing in the free-living bodonid Bodo saltans. Nucleic Acids Res 1998; 26:1205–1213 [View Article] [PubMed]
     [Google Scholar]
  13. Jackson AP, Otto TD, Aslett M, Armstrong SD, Bringaud F et al. Kinetoplastid phylogenomics reveals the evolutionary innovations associated with the origins of parasitism. Curr Biol 2016; 26:161–172 [View Article] [PubMed]
     [Google Scholar]
  14. Szokoli F, Castelli M, Sabaneyeva E, Schrallhammer M, Krenek S et al. Disentangling the taxonomy of rickettsiales and description of two novel symbionts (“Candidatus Bealeia paramacronuclearis” and “Candidatus Fokinia cryptica”) sharing the cytoplasm of the ciliate protist Paramecium biaurelia. Appl Environ Microbiol 2016; 82:7236–7247 [View Article] [PubMed]
     [Google Scholar]
  15. Castelli M, Petroni G. An evolutionary-focused review of the holosporales (Alphaproteobacteria): diversity, host interactions, and taxonomic re-ranking as holosporineae subord. Nov. Microb Ecol 2025; 88:15 [View Article] [PubMed]
     [Google Scholar]
  16. Schrallhammer M, Potekhin A. Epidemiology of Nucleus-Dwelling Holospora: Infection, Transmission, Adaptation, and Interaction with Paramecium. In Kloc M. eds Symbiosis: Cellular, Molecular, Medical and Evolutionary Aspects Cham: Springer International Publishing; 2020 pp 105–135 [View Article]
     [Google Scholar]
  17. Schrallhammer M, Castelli M, Petroni G. Phylogenetic relationships among endosymbiotic R-body producer: bacteria providing their host the killer trait. Syst Appl Microbiol 2018; 41:213–220 [View Article] [PubMed]
     [Google Scholar]
  18. Fujishima M. Infection and Maintenance of Holospora Species in Paramecium caudatum. In Fujishima M. eds Endosymbionts in Paramecium [Internet] vol 12 Berlin, Heidelberg: Springer Berlin Heidelberg; 2009 pp 201–225 [View Article]
     [Google Scholar]
  19. Fujishima M, Kawai M, Yamamoto R. Paramecium caudatum acquires heat-shock resistance in ciliary movement by infection with the endonuclear symbiotic bacterium Holospora obtusa. FEMS Microbiol Lett 2005; 243:101–105 [View Article] [PubMed]
     [Google Scholar]
  20. Midha S, Rigden DJ, Siozios S, Hurst GDD, Jackson AP. Bodo saltans (Kinetoplastida) is dependent on a novel Paracaedibacter-like endosymbiont that possesses multiple putative toxin-antitoxin systems. ISME J 2021; 15:1680–1694 [View Article] [PubMed]
     [Google Scholar]
  21. Jamet A, Nassif X. New players in the toxin field: polymorphic toxin systems in bacteria. mBio 2015; 6:e00285-15 [View Article] [PubMed]
     [Google Scholar]
  22. Macaulay IC, Haerty W, Kumar P, Li YI, Hu TX et al. G&T-seq: parallel sequencing of single-cell genomes and transcriptomes. Nat Methods 2015; 12:519–522 [View Article] [PubMed]
     [Google Scholar]
  23. Martin M. Cutadapt removes adapter sequences from high-throughput sequencing reads. EMBnet j 2011; 17:10 [View Article]
     [Google Scholar]
  24. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012; 19:455–477 [View Article] [PubMed]
     [Google Scholar]
  25. Kang DD, Li F, Kirton E, Thomas A, Egan R et al. MetaBAT 2: an adaptive binning algorithm for robust and efficient genome reconstruction from metagenome assemblies. PeerJ 2019; 7:e7359 [View Article] [PubMed]
     [Google Scholar]
  26. von Meijenfeldt FAB, Arkhipova K, Cambuy DD, Coutinho FH, Dutilh BE. Robust taxonomic classification of uncharted microbial sequences and bins with CAT and BAT. Genome Biol 2019; 20:217 [View Article]
     [Google Scholar]
  27. Laetsch DR, Blaxter ML. BlobTools: Interrogation of genome assemblies. F1000Res 2017; 6:1287 [View Article]
     [Google Scholar]
  28. Karlicki M, Antonowicz S, Karnkowska A. Tiara: deep learning-based classification system for eukaryotic sequences. Bioinformatics 2022; 38:344–350 [View Article]
     [Google Scholar]
  29. West PT, Probst AJ, Grigoriev IV, Thomas BC, Banfield JF. Genome-reconstruction for eukaryotes from complex natural microbial communities. Genome Res 2018; 28:569–580 [View Article]
     [Google Scholar]
  30. Parks DH, Imelfort M, Skennerton CT, Hugenholtz P, Tyson GW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res 2015; 25:1043–1055 [View Article] [PubMed]
     [Google Scholar]
  31. Haese-Hill W, Crouch K, Otto TD. Annotation and visualization of parasite, fungi and arthropod genomes with companion. Nucleic Acids Res 2024; 52:W39–W44 [View Article] [PubMed]
     [Google Scholar]
  32. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics 2014; 30:2068–2069 [View Article]
     [Google Scholar]
  33. Manni M, Berkeley MR, Seppey M, Simão FA, Zdobnov EM. BUSCO update: novel and streamlined workflows along with broader and deeper phylogenetic coverage for scoring of eukaryotic, prokaryotic, and viral genomes. Mol Biol Evol 2021; 38:4647–4654 [View Article] [PubMed]
     [Google Scholar]
  34. Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol 2013; 30:772–780 [View Article] [PubMed]
     [Google Scholar]
  35. Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol 2020; 37:1530–1534 [View Article]
     [Google Scholar]
  36. Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS. ModelFinder: fast model selection for accurate phylogenetic estimates. Nat Methods 2017; 14:587–589 [View Article] [PubMed]
     [Google Scholar]
  37. Letunic I, Bork P. Interactive Tree of Life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res 2024; 52:W78–W82 [View Article] [PubMed]
     [Google Scholar]
  38. Emms DM, Kelly S. OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biol 2019; 20:238 [View Article] [PubMed]
     [Google Scholar]
  39. Capella-Gutiérrez S, Silla-Martínez JM, Gabaldón T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics 2009; 25:1972–1973 [View Article] [PubMed]
     [Google Scholar]
  40. Borowiec ML. AMAS: a fast tool for alignment manipulation and computing of summary statistics. PeerJ 2016; 4:e1660 [View Article] [PubMed]
     [Google Scholar]
  41. Aramaki T, Blanc-Mathieu R, Endo H, Ohkubo K, Kanehisa M et al. KofamKOALA: KEGG Ortholog assignment based on profile HMM and adaptive score threshold. Bioinformatics 2020; 36:2251–2252 [View Article]
     [Google Scholar]
  42. Rodriguez-R LM, Konstantinidis KT. The enveomics collection: a toolbox for specialized analyses of microbial genomes and metagenomes. PeerJ Preprints 2016 [View Article]
     [Google Scholar]
  43. Gerhardt K, Ruiz-Perez CA, Rodriguez-R LM, Jain C, Tiedje JM et al. FastAAI: efficient estimation of genome average amino acid identity and phylum-level relationships using tetramers of universal proteins. Nucleic Acids Res 2025; 53:gkaf348 [View Article] [PubMed]
     [Google Scholar]
  44. Shaw J, Yu YW. Fast and robust metagenomic sequence comparison through sparse chaining with skani. Nat Methods 2023; 20:1661–1665 [View Article] [PubMed]
     [Google Scholar]
  45. Scheckenbach F, Wylezich C, Mylnikov AP, Weitere M, Arndt H. Molecular comparisons of freshwater and marine isolates of the same morphospecies of heterotrophic flagellates. Appl Environ Microbiol 2006; 72:6638–6643 [View Article] [PubMed]
     [Google Scholar]
  46. Schneider D, Zühlke D, Poehlein A, Riedel K, Daniel R. Metagenome-assembled genome sequences from different wastewater treatment stages in Germany. Microbiol Resour Announc 2021; 10:e0050421 [View Article] [PubMed]
     [Google Scholar]
  47. Muñoz-Gómez SA, Hess S, Burger G, Lang BF, Susko E et al. An updated phylogeny of the alphaproteobacteria reveals that the parasitic rickettsiales and holosporales have independent origins. eLife 2019; 8: [View Article]
     [Google Scholar]
  48. Parks DH, Rinke C, Chuvochina M, Chaumeil P-A, Woodcroft BJ et al. Recovery of nearly 8,000 metagenome-assembled genomes substantially expands the tree of life. Nat Microbiol 2017; 2:1533–1542 [View Article]
     [Google Scholar]
  49. Liu L, Wang Y, Yang Y, Wang D, Cheng SH et al. Charting the complexity of the activated sludge microbiome through a hybrid sequencing strategy. Microbiome 2021; 9:205 [View Article]
     [Google Scholar]
  50. Parks DH, Chuvochina M, Waite DW, Rinke C, Skarshewski A et al. A standardized bacterial taxonomy based on genome phylogeny substantially revises the tree of life. Nat Biotechnol 2018; 36:996–1004 [View Article]
     [Google Scholar]
  51. Probst AJ, Ladd B, Jarett JK, Geller-McGrath DE, Sieber CMK et al. Differential depth distribution of microbial function and putative symbionts through sediment-hosted aquifers in the deep terrestrial subsurface. Nat Microbiol 2018; 3:328–336 [View Article]
     [Google Scholar]
  52. Hess S, Suthaus A, Melkonian M. “Candidatus Finniella” (Rickettsiales, Alphaproteobacteria), novel endosymbionts of viridiraptorid amoeboflagellates (Cercozoa, Rhizaria). Appl Environ Microbiol 2016; 82:659–670 [View Article] [PubMed]
     [Google Scholar]
  53. Jain C, Rodriguez-R LM, Phillippy AM, Konstantinidis KT, Aluru S. High throughput ANI analysis of 90K prokaryotic genomes reveals clear species boundaries. Nat Commun 2018; 9:5114 [View Article] [PubMed]
     [Google Scholar]
  54. Goris J, Konstantinidis KT, Klappenbach JA, Coenye T, Vandamme P et al. DNA-DNA hybridization values and their relationship to whole-genome sequence similarities. Int J Syst Evol Microbiol 2007; 57:81–91 [View Article] [PubMed]
     [Google Scholar]
  55. Konstantinidis KT, Tiedje JM. Towards a genome-based taxonomy for prokaryotes. J Bacteriol 2005; 187:6258–6264 [View Article] [PubMed]
     [Google Scholar]
  56. Rodriguez-R LM, Konstantinidis KT. Bypassing cultivation to identify bacterial species: culture-independent genomic approaches identify credibly distinct clusters, avoid cultivation bias, and provide true insights into microbial species. Microbe Mag 2014; 9:111–118 [View Article]
     [Google Scholar]
  57. Park M-J, Kim YJ, Park M, Yu J, Namirimu T et al. Establishment of genome based criteria for classification of the family desulfovibrionaceae and proposal of two novel genera, Alkalidesulfovibrio gen. nov. and Salidesulfovibrio gen. nov. Front Microbiol 2022; 13:738205 [View Article] [PubMed]
     [Google Scholar]
  58. Harmer J, Yurchenko V, Nenarokova A, Lukeš J, Ginger ML. Farming, slaving and enslavement: histories of endosymbioses during kinetoplastid evolution. Parasitology 2018; 145:1311–1323 [View Article] [PubMed]
     [Google Scholar]
  59. Opperdoes FR, Butenko A, Flegontov P, Yurchenko V, Lukeš J. Comparative metabolism of free-living bodo saltans and parasitic trypanosomatids. J Eukaryot Microbiol 2016; 63:657–678 [View Article] [PubMed]
     [Google Scholar]
  60. Burki F, Sandin MM, Jamy M. Diversity and ecology of protists revealed by metabarcoding. Curr Biol 2021; 31:R1267–R1280 [View Article] [PubMed]
     [Google Scholar]
  61. Majda S, Boenigk J, Beisser D. Intraspecific variation in protists: clues for microevolution from Poteriospumella lacustris (Chrysophyceae). Genome Biol Evol 2019; 11:2492–2504 [View Article] [PubMed]
     [Google Scholar]
  62. Lachance MA, Lee DK, Hsiang T. Delineating yeast species with genome average nucleotide identity: a calibration of ANI with haplontic, heterothallic Metschnikowia species. Antonie Van Leeuwenhoek 2020; 113:2097–2106 [View Article] [PubMed]
     [Google Scholar]
  63. de Albuquerque NRM, Haag KL. Using average nucleotide identity (ANI) to evaluate microsporidia species boundaries based on their genetic relatedness. J Eukaryot Microbiol 2023; 70:e12944 [View Article] [PubMed]
     [Google Scholar]
  64. Konstantinidis KT, Tiedje JM. Prokaryotic taxonomy and phylogeny in the genomic era: advancements and challenges ahead. Curr Opin Microbiol 2007; 10:504–509 [View Article] [PubMed]
     [Google Scholar]
  65. Qin Q-L, Xie B-B, Zhang X-Y, Chen X-L, Zhou B-C et al. A proposed genus boundary for the prokaryotes based on genomic insights. J Bacteriol 2014; 196:2210–2215 [View Article] [PubMed]
     [Google Scholar]
  66. Makałowski W, Zhang J, Boguski MS. Comparative analysis of 1196 orthologous mouse and human full-length mRNA and protein sequences. Genome Res 1996; 6:846–857 [View Article] [PubMed]
     [Google Scholar]
  67. Wibberg D, Rupp O, Blom J, Jelonek L, Kröber M et al. Development of a rhizoctonia solani AG1-IB specific gene model enables comparative genome analyses between phytopathogenic R. solani AG1-IA, AG1-IB, AG3 and AG8 Isolates. PLoS One 2015; 10:e0144769 [View Article] [PubMed]
     [Google Scholar]
  68. Wibberg D, Stadler M, Lambert C, Bunk B, Spröer C et al. High quality genome sequences of thirteen Hypoxylaceae (Ascomycota) strengthen the phylogenetic family backbone and enable the discovery of new taxa. Fungal Diversity 2021; 106:7–28 [View Article]
     [Google Scholar]
  69. Groussin M, Mazel F, Alm EJ. Co-evolution and co-speciation of host-gut bacteria systems. Cell Host Microbe 2020; 28:12–22 [View Article] [PubMed]
     [Google Scholar]
  70. Russo L, Miller AD, Tooker J, Bjornstad ON, Shea K. Quantitative evolutionary patterns in bipartite networks: Vicariance, phylogenetic tracking or diffuse co‐evolution?. Methods Ecol Evol 2018; 9:761–772 [View Article]
     [Google Scholar]
  71. Schrallhammer M, Potekhin A. Epidemiology of Nucleus-Dwelling Holospora: Infection, Transmission, Adaptation, and Interaction with Paramecium. In Kloc M. eds Symbiosis: Cellular, Molecular, Medical and Evolutionary Aspects vol 69 Cham: Springer International Publishing; 2020 pp 105–135 [View Article]
     [Google Scholar]
  72. Frelier PF, Loy JK, Kruppenbach B. Transmission of necrotizing hepatopancreatitis in Penaeus vannamei. J Invertebr Pathol 1993; 61:44–48 [View Article]
     [Google Scholar]
  73. Schulz F, Lagkouvardos I, Wascher F, Aistleitner K, Kostanjšek R et al. Life in an unusual intracellular niche: a bacterial symbiont infecting the nucleus of amoebae. ISME J 2014; 8:1634–1644 [View Article] [PubMed]
     [Google Scholar]
  74. Korený L, Lukes J, Oborník M. Evolution of the haem synthetic pathway in kinetoplastid flagellates: an essential pathway that is not essential after all?. Int J Parasitol 2010; 40:149–156 [View Article] [PubMed]
     [Google Scholar]
  75. Alves JMP, Voegtly L, Matveyev AV, Lara AM, da Silva FM et al. Identification and phylogenetic analysis of heme synthesis genes in trypanosomatids and their bacterial endosymbionts. PLoS One 2011; 6:e23518 [View Article] [PubMed]
     [Google Scholar]
  76. Klein CC, Alves JMP, Serrano MG, Buck GA, Vasconcelos ATR et al. Biosynthesis of vitamins and cofactors in bacterium-harbouring trypanosomatids depends on the symbiotic association as revealed by genomic analyses. PLoS One 2013; 8:e79786 [View Article]
     [Google Scholar]
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Single-cell sequencing reveals unexpected genetic diversity among Bodo spp. flagellates and their bacterial endosymbionts
M Gen 12, 001642 (2026); https://doi.org/10.1099/mgen.0.001642
/content/journal/mgen/10.1099/mgen.0.001642
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