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Ciliates also have highly complex genomes which undergo extensive rearrangement during their development. Recently it was shown that in the genus Euplotes, the levels of synteny are low, see pmc.ncbi.nlm.nih.gov/articles/PMC...

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We base our orthology calls on careful protein sequence analysis, rather than on synteny. We did not check for synteny in this case, but we expect that considering the ancient divergence time from other sequenced species (>900 Mya from Paramecium) we are unlikely to find conservation of synteny.

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As a notable example, Thomas Cavalier-Smith has put out numerous different placements of the root based on gene presences/absences or molecular features, differing in conclusion depending on which feature was examined.

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Since you return to this criticism on sequence-based analyses, I would like to point out that analyses/hypotheses based on cell biological characteristics or molecular features have also yielded various different root positions.

That is what I meant to convey in my skeet. This is why robust and statistical sequence-based analyses are in our view much more informative. And these consistently group Euglenozoa together as a single, monophyletic group.

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This is precisely why we argue that basing the root on such characterics is not sustainable, as we explain in our paper. By placing the root within Euglenozoa, you appear to solve the diversity in kinetochore architecture, but this creates many other losses/transitions.

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However, I would like to address your point from your skeet 7/8. Of course we do not mean to say that morphologic changes do not happen in eukaryotic evolution. Eukaryotes come in all different shapes and sizes.

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Thank you for sharing your views. As you point out many of these points are discussed in our respective papers, so I'd like to refer to those and not go into all the details here.

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Highly divergent genes hiding in plain sight are definitely part of the puzzle! As an example, we recently identified numerous kinetochore components in the ciliate Tetrahymena thermophila that previously evaded sensitive homology searches: www.biorxiv.org/content/10.1...

biorxiv.org

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Finally, I'd like to thank my co-authors @jvhooff.bsky.social, Julius Lukeš, Thomas Richards, @andrewjroger.bsky.social, Bill Wickstead, @kopslab.bsky.social, Berend Snel & @eelcotromer.bsky.social for their valuable contributions and support. (12/12) #protistsonsky

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We are grateful to @jcellsci.bsky.social for providing the opportunity to express our views on this matter and for the smooth handling of our correspondence piece. (11/12)

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We conclude that the kinetoplastid kinetochore is the result of a replacement of an ancestral conventional kinetochore. This negates the idea that kinetoplastids branched off from all other eukaryotes directly after LECA and before the origin of the kinetochore. (10/12)

Genomic analysis finds no evidence of canonical eukaryotic DNA processing complexes in a free-living protist - Nature Communications

Furthermore, kinetoplastids are not the only known lineage to lack (almost) all components of the conventional kinetochore, as the metamonad Carpediemonas membranifera also was found to not encode many kinetochore subunits in its genome. See: www.nature.com/articles/s41... (9/12)

nature.com

The mechanisms for replicating and segregating DNA are highly conserved across eukaryotes. A comparative genomic analysis of a free-living protist finds a surprising lack of protein complexes involved...

Instead of a conventional kinetochore, kinetoplastids have an analogous system. In our reply, we point out that the replacement of core cell biological machinery by non-homologous proteins is known to have happened on multiple occasions across diverse eukaryotic lineages. (8/12)

By contrast, maintaining the monophyly of euglenids and kinetoplastids directly results in the inference of a conventional kinetochore in LECA, as euglenids possess numerous components of this system. Thus, the absence of a conventional kinetochore in kinetoplastids must be derived. (7/12)

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Unfortunately, @bungoakiyoshi.bsky.social responded to our reply without acknowledging these points. Instead, he argues that even in his proposed root placement, euglenids can be closely-related to kinetoplastids, despite their paraphyly. We stress that these notions are mutually exclusive.(6/12)

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Placing the LECA root within Euglenozoa, as @bungoakiyoshi.bsky.social proposes, is problematic because it implies that LECA itself was a Euglenozoan-like cell. This scenario necessitates the loss of many Euglenozoa-specific features in the branch leading to all other eukaryotes. (5/12)

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Critically, the proposed scenario would mean that euglenids are more closely-related to all other eukaryotes, including humans, than they are to kinetoplastids. This is dubious as there is a lot of evidence showing the close relation between euglenids and kinetoplastids, together Euglenozoa. (4/12)

This scenario places the root of the eukaryotic tree of life between kinetoplastids and all other eukaryotes. However, no phylogenetic support exists for this idea. (3/12)

In their Hypothesis, @bungoakiyoshi.bsky.social interprets the fact that kinetoplastids lack a conventional kinetochore, a core machinery for cell division, as evidence for these organisms having split-off before the emergence of the conventional kinetochore system. (2/12)

The LECA had a conventional kinetochore and the kinetoplastid kinetochore is a derived feature – a critical evaluation of Akiyoshi, 2025

Recently, a Hypothesis was posed in @jcellsci.bsky.social in which the root of eukaryotes was placed between kinetoplastids and all other eukaryotes. From this, it was implied that LECA did not have a kinetochore. We argue this is highly unlikely. A 🧵(1/12) Read our reply here: tinyurl.com/n87myhpr

tinyurl.com

Summary:Akiyoshi, 2025 presented a hypothesis with implications for the early evolution of eukaryotes and eukaryotic cell division machinery. In this Correspondence, the authors conclude that this hyp...

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This work would not have been possible without Emine Ali, my co-first author and resident Tetrahymena expert, and co-authors @lauraelse.bsky.social , Harmjan, Paula, and of course my supervisors @eelcotromer.bsky.social, Berend Snel and @kopslab.bsky.social. Thanks to all! (11/11)

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All in all, we find that T. thermophila has a unique kinetochore combining both highly-divergent, but ancient, as well as more recently-evolved components into a functional whole. (10/11)

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Finally, we identified one unconventional component to be a highly-divergent member of the kinesin-6 family. So far, no kinesin-6 family member has been reported at the kinetochore in model organisms, but its presence in T. thermophila may suggest an ancestral function here. (9/11)

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We find three unconventional components localising to the inner kinetochore, a known hub for evolutionary novelty. Our homology searches revealed that they have very diverse origins despite likely co-orchestrating the various functions of the inner kinetochore. (8/11)

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To uncover how the different components assemble in the T. thermophila kinetochore, we performed nanometer-scale intra-kinetochore measurements. This revealed that the T. thermophila kinetochore assembles much like the human kinetochore despite high levels of divergence. (7/11)

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There were also proteins without homology to known kinetochore components. Further searches revealed that they have a wide range of evolutionary backgrounds, some neofunctionalised, others arose through lineage-specific gene birth and yet others may be unrecognised ancestral kinetochore units.(6/11)

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Surprisingly, we found no orthologs of either the Dam1 nor the Ska complexes, and also a large number of spindle assembly checkpoint proteins remained elusive. (5/11)

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Using deep-homology searches with sequence-based methods as well as AlphaFold-based structural searches, we found a set of highly-diverse orthologs of known kinetochore components. Combined, these constitute a primary axis of the kinetochore: CENP-C, the MIS12 complex and the NDC80 complex. (4/11)

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By performing proximity-labeling proteomics combined with microscopy on the few known kinetochore components in the classic model organism T. thermophila, we uncovered a range of proteins associated to its kinetochore, but it was unclear what the evolutionary origins of these proteins were. (3/11)

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The kinetochore composition of ciliates has long been elusive, as they seemed to lack many known components. Moreover, they have a unique cell division process, as are binucleated but only one undergoes mitosis. We wanted to find out how they segregate their chromosomes during cell division. (2/11)