Phylogenomics of free-living neobodonids reveals they are a paraphyletic group from which all other metakinetoplastids are descended
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eLife Assessment
This valuable study substantially improves the phylogenetic framework for Kinetoplastea through greatly expanded sampling of free-living neobodonids; the phylogenomic analyses provide convincing evidence that neobodonids are paraphyletic and establish a substantially revised framework for deep relationships within Metakinetoplastina. The work forms the foundation for future comparative studies of kinetoplastid evolution and systematics, although its implications for the origins of parasitism should not be overstated. An 18S rRNA phylogeny documenting the placement of the newly sampled strains within previously recognized neobodonid lineages and/or confirmation of the principal topology using a well-documented phylogenomic set of marker genes would further strengthen the study. The work will be of particular interest to researchers studying protist evolution and the evolutionary context of trypanosomatids.
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Abstract
Kinetoplastea is a major taxon of microbial eukaryotes that includes the well-known trypanosomatid parasites, species of which cause diseases in humans, animals and plants. Free-living kinetoplastids are greatly understudied compared to their parasitic relatives, but are ecologically important microbivores, and collectively comprise the great majority of kinetoplastid diversity. For two decades kinetoplastids have been divided into prokinetoplastids and metakinetoplastids, with the latter further split into four orders, the most diverse of which is Neobodonida. However, the position of the root of the metakinetoplastids and whether neobodonids are a clade has remained unclear due to very limited multi-gene data from free-living kinetoplastids, particularly neobodonids. Here, we present transcriptomic data for eleven newly or recently cultivated free-living neobodonid kinetoplastids. Phylogenomic analyses of a de novo generated data set of 444 inferred orthologs and 49 taxa robustly resolve the kinetoplastid tree, including the position of the root of metakinetoplastids. This divides metakinetoplastids such that Trypanosomatida, Eubodonida, Parabodonida, Allobodonidae (formerly neobodonid clade 1E) and neobodonid clade 1D fall on one side, while neobodonid clades Nd6, 1B (Rhynchomonadidae) and 1C fall on the other. Neobodonids are thus inferred to be a paraphyletic group from which all other metakinetoplastids descend. This analysis is the most thorough examination of metakinetoplastid phylogeny thus far, and forms a new basis for tracing the evolutionary history of the entire kinetoplastid group.
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eLife Assessment
This valuable study substantially improves the phylogenetic framework for Kinetoplastea through greatly expanded sampling of free-living neobodonids; the phylogenomic analyses provide convincing evidence that neobodonids are paraphyletic and establish a substantially revised framework for deep relationships within Metakinetoplastina. The work forms the foundation for future comparative studies of kinetoplastid evolution and systematics, although its implications for the origins of parasitism should not be overstated. An 18S rRNA phylogeny documenting the placement of the newly sampled strains within previously recognized neobodonid lineages and/or confirmation of the principal topology using a well-documented phylogenomic set of marker genes would further strengthen the study. The work will be of particular interest to …
eLife Assessment
This valuable study substantially improves the phylogenetic framework for Kinetoplastea through greatly expanded sampling of free-living neobodonids; the phylogenomic analyses provide convincing evidence that neobodonids are paraphyletic and establish a substantially revised framework for deep relationships within Metakinetoplastina. The work forms the foundation for future comparative studies of kinetoplastid evolution and systematics, although its implications for the origins of parasitism should not be overstated. An 18S rRNA phylogeny documenting the placement of the newly sampled strains within previously recognized neobodonid lineages and/or confirmation of the principal topology using a well-documented phylogenomic set of marker genes would further strengthen the study. The work will be of particular interest to researchers studying protist evolution and the evolutionary context of trypanosomatids.
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Reviewer #1 (Public review):
Summary:
The authors present the most comprehensive phylogenomic study of free-living kinetoplastids to date, including newly generated transcriptomic data from 11 species. This dataset provides a solid foundation for future comparative evolutionary studies of kinetoplastids, a group that includes important human pathogens such as Trypanosoma brucei.
Strengths:
The study is clearly written, and the analyses are thoroughly described, well documented, and reproducible. The phylogenomic dataset comprises 444 proteins, which is larger than those used in many comparable phylogenomic studies. The main strength of the study is the substantially improved taxon sampling of neobodonids, which provides a much stronger framework for resolving evolutionary relationships among free-living kinetoplastids.
Weaknesses:
The …
Reviewer #1 (Public review):
Summary:
The authors present the most comprehensive phylogenomic study of free-living kinetoplastids to date, including newly generated transcriptomic data from 11 species. This dataset provides a solid foundation for future comparative evolutionary studies of kinetoplastids, a group that includes important human pathogens such as Trypanosoma brucei.
Strengths:
The study is clearly written, and the analyses are thoroughly described, well documented, and reproducible. The phylogenomic dataset comprises 444 proteins, which is larger than those used in many comparable phylogenomic studies. The main strength of the study is the substantially improved taxon sampling of neobodonids, which provides a much stronger framework for resolving evolutionary relationships among free-living kinetoplastids.
Weaknesses:
The phylogenomic analysis is based on a newly assembled dataset comprising 444 single-copy orthogroups. Using an established phylogenomic dataset, expanded to include the newly studied species, to confirm the inferred relationships would provide additional support for the robustness of the recovered topology.
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Reviewer #2 (Public review):
Summary:
This is a highly competent and very throough phylogenomic study of the position of neobodonids within the Kinetoplastid phylogeny. The addition of new transcriptomic data for diverse neobodonids allows their relationships to be better understood, leading to convincing changes to neobodonid taxonomy.
Strengths:
The generation of transcriptomic data produces more phylogenetic characters than ever before from several, diverse neobodonid lineages. This represents a substantial increase in the data available for this relatively unstudied group. The methodology used to define the character set and then estimate the phylogeny is conventional, very thoroughly described and expertly executed. The phylogeny is highly robust, and we can be very confident of the result.
Weaknesses:
These changes to neobodonid …
Reviewer #2 (Public review):
Summary:
This is a highly competent and very throough phylogenomic study of the position of neobodonids within the Kinetoplastid phylogeny. The addition of new transcriptomic data for diverse neobodonids allows their relationships to be better understood, leading to convincing changes to neobodonid taxonomy.
Strengths:
The generation of transcriptomic data produces more phylogenetic characters than ever before from several, diverse neobodonid lineages. This represents a substantial increase in the data available for this relatively unstudied group. The methodology used to define the character set and then estimate the phylogeny is conventional, very thoroughly described and expertly executed. The phylogeny is highly robust, and we can be very confident of the result.
Weaknesses:
These changes to neobodonid taxonomy, or essentially the discovery that some 'neobodonids' actually represent distinct lineages at the base of the Metakinetoplastida, are not surprising. So undersampled are the Kinetoplastida, both in terms of molecular markers and phyletic diversity, that an investment in sampling on this scale is always likely to bring about changes. The significance of these changes is not immediately apparent. Since they do not concern the Eubodonidae, they have no implications for the trypanosomatids and the new Metakinetoplastid lineages are very distant from the origins of parasitism, such that comments on "preadaptations for disease" are not compelling.
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Reviewer #3 (Public review):
Summary:
The manuscript by Zavadska et al. presents a phylogenomic study focused on the relationships among metakinetoplastids, specifically addressing the question whether Neobodonida are monophyletic. The authors collected eleven kinetoplastid strains affiliated with five out of six neobodonid clades identified in previous studies and sequenced their transcriptomes. These data along with SRAs available from public databases were processed via assembly, annotation and decontamination. The final dataset, representing all (non-trypanosomatid) kinetoplastids with genome and/or transcriptome data available as well as selected trypanosomatids and discoban outgroups, was used for protein orthology inference and identification of single-copy orthologs present in the majority of taxa, followed by phylogenetic …
Reviewer #3 (Public review):
Summary:
The manuscript by Zavadska et al. presents a phylogenomic study focused on the relationships among metakinetoplastids, specifically addressing the question whether Neobodonida are monophyletic. The authors collected eleven kinetoplastid strains affiliated with five out of six neobodonid clades identified in previous studies and sequenced their transcriptomes. These data along with SRAs available from public databases were processed via assembly, annotation and decontamination. The final dataset, representing all (non-trypanosomatid) kinetoplastids with genome and/or transcriptome data available as well as selected trypanosomatids and discoban outgroups, was used for protein orthology inference and identification of single-copy orthologs present in the majority of taxa, followed by phylogenetic analysis in maximum likelihood and Bayesian frameworks.
Strengths:
I find the new data and resulting phylogenetic reconstruction valuable and interesting, particularly given the paucity of genomic/transcriptomic data from free-living kinetoplastids. The study is well-designed methodologically, and the main finding (paraphyly of Neobodonida) appears indisputable, considering the provided phylogenimic evidence. Following best practices, the authors shared their primary data, scripts, and files resulting from the conducted analyses.
Weaknesses:
Several aspects of the presentation and interpretation would benefit from revision. In particular, the composition of the neobodonid lineages central to the study is insufficiently explained, and an 18S rRNA gene-based tree showing these lineages and the placement of the newly analysed strains would substantially improve the clarity of the study. The Introduction would also benefit from a more concise and better-structured account of previous phylogenetic evidence and a clearer explanation of the broader biological significance of kinetoplastids, while the Methods could be streamlined by removing redundant information and presenting some details in tables. Finally, the rationale for including the electron microscopy images in the supplement should be clarified, as these are currently not discussed in the text and represent only a single studied strain.
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Author response:
Reviewer #1 (Public review):
Summary:
The authors present the most comprehensive phylogenomic study of free-living kinetoplastids to date, including newly generated transcriptomic data from 11 species. This dataset provides a solid foundation for future comparative evolutionary studies of kinetoplastids, a group that includes important human pathogens such as Trypanosoma brucei.
Strengths:
The study is clearly written, and the analyses are thoroughly described, well documented, and reproducible. The phylogenomic dataset comprises 444 proteins, which is larger than those used in many comparable phylogenomic studies. The main strength of the study is the substantially improved taxon sampling of neobodonids, which provides a much stronger framework for resolving evolutionary relationships among free-living …
Author response:
Reviewer #1 (Public review):
Summary:
The authors present the most comprehensive phylogenomic study of free-living kinetoplastids to date, including newly generated transcriptomic data from 11 species. This dataset provides a solid foundation for future comparative evolutionary studies of kinetoplastids, a group that includes important human pathogens such as Trypanosoma brucei.
Strengths:
The study is clearly written, and the analyses are thoroughly described, well documented, and reproducible. The phylogenomic dataset comprises 444 proteins, which is larger than those used in many comparable phylogenomic studies. The main strength of the study is the substantially improved taxon sampling of neobodonids, which provides a much stronger framework for resolving evolutionary relationships among free-living kinetoplastids.
Weaknesses:
The phylogenomic analysis is based on a newly assembled dataset comprising 444 single-copy orthogroups. Using an established phylogenomic dataset, expanded to include the newly studied species, to confirm the inferred relationships would provide additional support for the robustness of the recovered topology.
We thank the reviewer for their comments on our work. We appreciate the suggestion to confirm the topology that we recovered by using an established phylogenomic dataset. We note that we did not choose to use an established dataset for the main phylogenomic analysis, as we found that free-living kinetoplastid orthologs are systematically excluded from commonly used “universal pan-eukaryotic” datasets (e.g., Fig. S4), despite the fact that we were able to identify sufficient orthologous proteins to robustly reconstruct interspecies relationships among euglenozoans. In our revised manuscript, we will implement the reviewer’s suggestion by comparing our orthogroup set with commonly used pan-eukaryotic orthogroup sets.
Reviewer #2 (Public review):
Summary:
This is a highly competent and very throough phylogenomic study of the position of neobodonids within the Kinetoplastid phylogeny. The addition of new transcriptomic data for diverse neobodonids allows their relationships to be better understood, leading to convincing changes to neobodonid taxonomy.
Strengths:
The generation of transcriptomic data produces more phylogenetic characters than ever before from several, diverse neobodonid lineages. This represents a substantial increase in the data available for this relatively unstudied group. The methodology used to define the character set and then estimate the phylogeny is conventional, very thoroughly described and expertly executed. The phylogeny is highly robust, and we can be very confident of the result.
Weaknesses:
These changes to neobodonid taxonomy, or essentially the discovery that some 'neobodonids' actually represent distinct lineages at the base of the Metakinetoplastida, are not surprising. So undersampled are the Kinetoplastida, both in terms of molecular markers and phyletic diversity, that an investment in sampling on this scale is always likely to bring about changes. The significance of these changes is not immediately apparent. Since they do not concern the Eubodonidae, they have no implications for the trypanosomatids and the new Metakinetoplastid lineages are very distant from the origins of parasitism, such that comments on "preadaptations for disease" are not compelling.
We agree that comments and discussion on how our study may shed light on the preadaptations of trypanosomatids to parasitism were excessive. We were influenced by prior studies of free-living metakinetoplastids, which highlighted their importance and applied value in the context of understanding evolution of parasitism in kinetoplastids (Deschamps et al., 2011; Dolezel et al., 2000; Flegontova et al., 2017). In our revised manuscript, we will remove/shorten the corresponding parts of discussion and results, and place more emphasis on the relevance of our study to understand the multiple origins of parasitism in metakinetoplastids, not just trypanosomatids.
The datasets published in our study can be used in the future to investigate, for example, how gene repertoire of trypanosomatids differs from the free-living species, or how specific gene families evolved, and whether their diversification/origin predated the origin of the largest parasitic clade. We acknowledge that the data from the closest free-living relative of trypanosomatids that was available before our study is already extremely valuable, and that our datasets would be of most use for reconstructing relatively ancient preadaptations.
Reviewer #3 (Public review):
Summary:
The manuscript by Zavadska et al. presents a phylogenomic study focused on the relationships among metakinetoplastids, specifically addressing the question whether Neobodonida are monophyletic. The authors collected eleven kinetoplastid strains affiliated with five out of six neobodonid clades identified in previous studies and sequenced their transcriptomes. These data along with SRAs available from public databases were processed via assembly, annotation and decontamination. The final dataset, representing all (non-trypanosomatid) kinetoplastids with genome and/or transcriptome data available as well as selected trypanosomatids and discoban outgroups, was used for protein orthology inference and identification of single-copy orthologs present in the majority of taxa, followed by phylogenetic analysis in maximum likelihood and Bayesian frameworks.
Strengths:
I find the new data and resulting phylogenetic reconstruction valuable and interesting, particularly given the paucity of genomic/transcriptomic data from free-living kinetoplastids. The study is well-designed methodologically, and the main finding (paraphyly of Neobodonida) appears indisputable, considering the provided phylogenimic evidence. Following best practices, the authors shared their primary data, scripts, and files resulting from the conducted analyses.
Weaknesses:
Several aspects of the presentation and interpretation would benefit from revision. In particular, the composition of the neobodonid lineages central to the study is insufficiently explained, and an 18S rRNA gene-based tree showing these lineages and the placement of the newly analysed strains would substantially improve the clarity of the study. The Introduction would also benefit from a more concise and better-structured account of previous phylogenetic evidence and a clearer explanation of the broader biological significance of kinetoplastids, while the Methods could be streamlined by removing redundant information and presenting some details in tables. Finally, the rationale for including the electron microscopy images in the supplement should be clarified, as these are currently not discussed in the text and represent only a single studied strain.
We thank the reviewer for their suggestions on how our manuscript could be improved. Regarding the selection of species, our study followed a common procedure in protistology: we attempted to isolate, culture and sequence as many free-living kinetoplastids as possible, and the resulting manuscript includes all the cases in which we were successful. We appreciate the reviewer’s suggestion of including an 18S rRNA gene tree, which we will implement in our revised manuscript. We will revise the introduction to improve the presentation of previous phylogenetic evidence in metakinetoplastids and to provide a more clear explanation of their broader significance. We also plan to reduce redundant information in the Methods and relocate information on strains and culturing conditions to tables.
Electron microscopy images in the Supplement display the ultrastructure of an isolate from the 1D clade. They were included because the information on morphology of this clade remains scarce in comparison to other clades; thus, we added these images because we believe they could be useful for the community. We intend to clarify this in our revised manuscript.
References
Deschamps P, Lara E, Marande W, López-García P, Ekelund F, Moreira D (2011) Phylogenomic analysis of kinetoplastids supports that trypanosomatids arose from within bodonids Mol Biol Evol 28:53-58 https://doi.org/10.1093/molbev/msq289
Dolezel D, Jirků M, Maslov DA, Lukes J (2000) Phylogeny of the bodonid flagellates (Kinetoplastida) based on small-subunit rRNA gene sequences Int J Syst Evol Microbiol 50:1943-1951 https://doi.org/10.1099/00207713-50-5-1943
Flegontova O, Flegontov P, Malviya S, Poulain J, de Vargas C, Bowler C, Lukeš J, Horák A (2018) Neobodonids are dominant kinetoplastids in the global ocean Environ Microbiol 20:878-889 https://doi.org/10.1111/1462-2920.14034
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