# Repeated duplications and losses shaped SMC complex evolution from archaeal ancestors to modern eukaryotes

Jolien J.E. van Hooff, Maximilian W.D. Raas, Eelco Tromer, Laura Eme

► To cite this version:

Jolien J.E. van Hooff, Maximilian W.D. Raas, Eelco Tromer, Laura Eme. Repeated duplications and losses shaped SMC complex evolution from archaeal ancestors to modern eukaryotes. Cell Reports, 2025, 44 (7), pp.115855. 10.1016/j CELREP.2025.115855 hal-05404770

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**Cell Reports**

# Repeated duplications and losses shaped SMC complex evolution from archaeal ancestors to modern eukaryotes

## Graphical abstract

<figure><img src="image_2.png" /></figure>

## Authors

Jolien J.E. van Hooff, Maximilian W. D. Raas, Eelco C. Tromer, Laura Eme

## Correspondence

jolien.vanhooff@wur.nl (J.J.E.v.H.), laura.eme@uri.edu (L.E.)

## In brief

Van Hooff et al. reconstruct the evolutionary histories of eukaryotic SMC complexes, molecular machineries that organize chromosomes. SMC complexes were fully present in the last eukaryotic common ancestor (LECA) but have been frequently lost during evolution. Remarkably, these complexes originated from an ancient duplication in archaeal ancestors, suggesting that sophisticated chromosome organization predated eukaryotes.

## Highlights

* The last eukaryotic common ancestor had all four modern SMC complexes

* Condensin II was lost over 30 times during eukaryotic evolution

* Multiple SMC complex components are more ancient than previously appreciated

* A single SMC complex was duplicated in an ancient archaeal ancestor of eukaryotes

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Article

# Repeated duplications and losses shaped SMC complex evolution from archaeal ancestors to modern eukaryotes

Jolien J.E. van Hooff,<sup>1,2,8,*</sup> Maximilian W.D. Raas,<sup>3,4,5</sup> Eelco C. Tromer,<sup>6</sup> and Laura Eme<sup>1,7,*</sup>

## SUMMARY

Across life, structural maintenance of chromosomes (SMC) complexes organize chromosomes. While most prokaryotes have one, eukaryotes usually possess four (condensin I, condensin II, cohesin, and SMC5/6), shaping their considerably larger genomes. Although essential, SMC complexes differ among model eukaryotes, suggesting underexplored diversity. Here, we reconstruct eukaryotic SMC complex evolution, revealing that the last eukaryotic common ancestor (LECA) had all four complexes, supporting a sophisticated LECA. Subsequently, condensin II was lost at least 30 times, making it one of the most frequently lost eukaryotic machineries. Moreover, multiple SMC complex components are more ancient and widespread than previously appreciated. Tracing prokaryotic origins, we propose that the SMC complex was already duplicated in the Thaumarchaeota-Aigarchaeota-Crenarchaeota-Korarchaeota (TACK) and Asgard archaeal ancestor, suggesting sophisticated chromosome organization in eukaryotes' archaeal ancestor. Gene duplications further expanded the eukaryotic SMC complex inventory, highlighting their significance in establishing eukaryotic complexity. Altogether, our work suggests major shifts in genome organization throughout eukaryote history.

## INTRODUCTION

The organization of large eukaryotic nuclear genomes is a complex and dynamic process.<sup>1</sup> At the lowest level, DNA is wrapped around a histone octamer, forming a nucleosome (Figure 1). The DNA then forms topologically associating domains (TADs), wherein DNA sequences interact more frequently with each other than with other regions, with consequences for the regulation of gene expression.<sup>1</sup> At a higher organizational level, chromosomes consist of two main types of spatially distinct compartments, termed “A” (transcriptionally active euchromatin) and “B” (silenced heterochromatin) compartments. At the highest structural level, chromosomes typically occupy their own region in the nucleus. To ensure efficient and faithful DNA replication, maintaining genome stability and minimizing DNA damage, and to allow intricate regulation

of gene expression, these organizational levels need to be carefully coordinated.

SMC (structural maintenance of chromosomes) complexes have versatile and crucial roles in regulating these different levels. SMC complexes interact with DNA by hydrolyzing ATP. This drives a conformational change that allows them to capture DNA and then move it through the complex. With that, they build and progressively enlarge DNA loops, a process referred to as loop extrusion.<sup>2</sup> This ability underlies many of the functions of SMC complexes, facilitating the spatial and functional organization of the genome.

SMC complexes have been identified in all three domains of life<sup>3</sup> and have been shown to function in chromosome condensation, genetic recombination, and DNA replication and repair. In addition, in eukaryotes, they are responsible for sister chromatid cohesion.<sup>4</sup> Eukaryotes typically possess

<figure><img src="image_4.png" /></figure>

<footnote n="1"><sup>1</sup>Unité d'Ecologie Systématique et Evolution, CNRS, Université Paris-Saclay, AgroParisTech, Site IDEEV 12, Route 128, 91190 Gif-sur-Yvette, France</footnote>

<footnote n="2"><sup>2</sup>Laboratory of Microbiology, Wageningen University and Research, Helix (Building 124), Stippeneng 4, 6708WE Wageningen, the Netherlands</footnote>

<footnote n="3"><sup>3</sup>Theoretical Biology and Bioinformatics, Faculty of Science, Utrecht University, Padualaan 8, 3584CH Utrecht, the Netherlands</footnote>

<footnote n="4"><sup>4</sup>Hubrecht Institute, Royal Netherlands Academy of Arts and Sciences (KNAW), Uppsalalaan 8, 3584CT Utrecht, the Netherlands</footnote>

<footnote n="5"><sup>5</sup>Oncode Institute, Jaarbeursplein 6, 3521AL Utrecht, the Netherlands</footnote>

<footnote n="6"><sup>6</sup>Cell Biochemistry, Groningen Biomolecular Sciences & Biotechnology Institute, University of Groningen, Linneausborg, Nijenborgh 7, 9747AG Groningen, the Netherlands</footnote>

<footnote n="7"><sup>7</sup>Department of Cell and Molecular Biology, Center for Biotechnology and Life Sciences, University of Rhode Island, 120 Flagg Road, Kingston, RI 02881, USA</footnote>

<footnote n="8"><sup>8</sup>Lead contact</footnote>

<footnote>*Correspondence: jolien.vanhooff@wur.nl (J.J.E.v.H.), laura.eme@uri.edu (L.E.)</footnote>

<footnote>https://doi.org/10.1016/j.celrep.2025.115855</footnote>
<figure><img src="image_5.png" /><figcaption>**Figure 1. Schematic of hierarchical nuclear genome organization in eukaryotes**</figcaption></figure>

The typical structural unit of eukaryotic genome organization is the nucleosome, in which 147 bp surround a histone octamer. Longer stretches of DNA form TADs, regions exhibiting higher interaction frequencies within them compared to adjacent regions. At a higher level, chromosomes are partitioned into distinct A and B compartments. A compartments are enriched with transcriptionally active euchromatin, while B compartments contain repressed heterochromatin. Finally, individual chromosomes are confined to specific subspaces within the nucleus, known as chromosome territories. Created with BioRender.

four SMC complexes (condensin I, condensin II, cohesin, and SMC5/6), whereas most prokaryotes have just one (Figure 2B). All SMC complexes share a common basic structure, consisting of two SMC proteins that dimerize via their hinge domains to form a composite ATPase (Figure 2A). A long and flexible kleisin protein binds asymmetrically to one SMC protein's neck and to the other's cap, resulting in a tripartite ring that is supplemented with different proteins, depending on the complex type. Eukaryotic condensin I and II and cohesin contain Huntingtin-Elongation factor 3-regulatory subunit A of protein phosphatase 2A-Target of rapamycin (HEAT) protein associated with a kleisin (hawk) proteins, while prokaryotic SMC complexes and the eukaryotic SMC5/6 complex contain kleisin-interacting tandem winged-helix element (kite) proteins. Hawks and kites do not appear to be homologous.⁵ Eukaryotic SMC complexes also incorporate accessory, sometimes transient, subunits, which regulate their activity and localization.

Each complex has distinct roles, but there are areas of overlap, and several functions remain unclear. Condensin I and II complexes, which condense chromosomes during mitosis,⁶ are composed of identical SMC proteins (SMC2 and SMC4) but differ in their kleisin (CAPH versus CAPH2) and hawk proteins (CAPG and CAPD2 versus CAPG2 and CAPD3, respectively) (Figures 2A and 3). The impact of condensin II extends to interphase, as it induces the formation of chromosome territories.⁷ This appears to be consistent across studied species, as those lacking condensin II do not have territories. Instead, they exhibit a distinct nuclear arrangement known as a “Rabl”-like architecture, characterized by the clustering of centromeres and/or telomeres, and often their attachment to the nuclear envelope.⁸

The primary role of cohesin is in maintaining sister chromatids in cohesion from DNA replication until anaphase, ensuring that chromosomes are properly aligned and attached to the mitotic spindle for safe segregation. Furthermore, at least in vertebrates, cohesin organizes DNA into loops, which, in turn, form TADs. Cohesin's composition seems to be more intricate than that of condensin, characterized by a diverse array of core and peripheral subunits (Figure 3).

Finally, the SMC5/6 complex stands out as the most cryptic eukaryotic SMC complex. In addition to the core elements (Figures 2A and 3), it contains a small ubiquitin-like modifier (SUMO) ligase (Nse2) and an α helix-rich subcomplex (Nse5 and Nse6).⁹ The SMC5/6 complex is implicated in a variety of cellular processes, including genome replication, DNA damage repair, and defense against viral DNA.⁹

The absence of some SMC complexes has been reported in distantly related eukaryotic lineages,¹⁰⁻¹² but their precise distribution and composition, evolutionary history during the diversification of eukaryotes, and impact on genome organization in non-model organisms remain unknown. In addition, the evolutionary events leading to the expansion of the SMC complex repertoire in eukaryotes remain to be precisely determined. Yoshinaga and Inagaki suggested that two SMC complexes were already present in the Asgard archaeal ancestor of eukaryotes,¹³⁻¹⁵ which, later, in early eukaryotic evolution, gave rise to condensin plus cohesin and to SMC5/6, respectively.¹⁰ However, the sequence of evolutionary events leading to the emergence of the four eukaryotic SMC complexes and their distinct subunits remains unresolved. Specifically, the evolution of non-SMC protein components, including the kleisin, kite, and hawk families, has not yet been studied. As a result, it is not yet known whether the archaeal ancestor of eukaryotes indeed possessed two distinct, complete SMC complexes.

Here, we systematically investigate the distribution and evolution of eukaryotic SMC complexes both before and after the last eukaryotic common ancestor (LECA). We reconstruct the SMC repertoire of LECA, uncover how these complexes diversified post-LECA, and propose a scenario for the origin of all four SMC complexes pre-LECA. Our analysis is based on state-of-the-art homology detection, including structural comparisons, and molecular phylogenetics, applied to a diverse dataset of 177 eukaryotic and 4,101 prokaryotic proteomes, including 28 Asgard archaea—the closest extant prokaryotic relatives of eukaryotes.¹⁵

Our findings shed light on the evolution of these hallmark eukaryotic protein complexes, provide insights into genome
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /><figcaption>**Figure 2. SMC complex composition and evolution**</figcaption></figure>

(A) General composition and architecture of SMC complexes across the tree of life. The SMC proteins form dimers via their hinge domains (top), and their ATPase heads are linked to the kleisin protein (bottom). Between the head and hinge domains, they feature a backfolded coiled-coil region. The kleisin attaches to one SMC protein through its N terminus, binding the SMC protein at its neck (v-SMC) and to the other SMC protein via its C terminus, binding it at the SMC protein's cap (κ-SMC). Note that, in prokaryotic SMC complexes, v-SMC and κ-SMC are the same protein, forming a homodimer. In eukaryotic SMC complexes, these are different proteins forming a heterodimer. In addition to the SMC and kleisin proteins, SMC complexes generally include two other core subunits, either hawk proteins (the eukaryotic condensin and cohesin complexes) or kite proteins (prokaryotic SMC complexes and the eukaryotic SMC5/6 complex). In addition to the displayed subunits, eukaryotic SMC complexes often have additional accessory members and/or more transient interactors (Figure 3).

(B) Illustration of the tree of life with this study's research questions. Eukaryotes typically have four SMC complexes: condensin I, condensin II, cohesin, and SMC5/6. Research questions are as follows. (1) What is the diversity of SMC complexes among eukaryotes? (2) What were the compositions of the SMC complexes in the last eukaryotic common ancestor (LECA)? (3) How did eukaryotic SMC complexes and their subunits originate before LECA? Created with BioRender.

organization in non-model eukaryotes, and offer a valuable framework for in-depth functional investigation of individual proteins through their sequence evolution.

## RESULTS

### Condensin II was lost recurrently during eukaryotic evolution

We first searched for orthologs of condensin I and II subunits across a diverse range of eukaryotes (STAR Methods). We confirmed that SMC2 and SMC4 are widely conserved, suggesting that the vast majority of species possess at least one condensin complex (Figure 3; Table S1). Our analysis revealed that the subunits specific to each complex (i.e., CAPH, CAPG, and CAPD2 [condensin I] and CAPH2, CAPG2, and CAPD3 [condensin II]) are generally coherently present or absent, suggesting their co-evolution across eukaryotic diversity.

The absence of complex-specific subunits in various eukaryotic species suggests multiple independent losses of condensin I and II (at least 6 and 30 losses, respectively). To avoid false negatives owing to extensive sequence divergence or incomplete data, we only considered a condensin complex absent from a given lineage if all three complex-specific proteins were undetected. Accordingly, condensin II subunits are among the most frequently lost proteins in eukaryotic evolution,¹⁶⁻¹⁹ matching

several kinetochore proteins²⁰ and a cilium-associated small GTPase.²¹ The absence of condensin II has been reported to correlate with a Rab-like architecture¹² (although exceptions exist; Discussion), and the remarkably high frequency of condensin II losses suggests that transitions from chromosome territories to Rab-like interphase nuclear architectures were common during eukaryotic evolution. Moreover, it suggests that, in contrast to condensin I, condensin II is often dispensable for condensation of chromosomes during mitosis.

Conversely, we find that certain lineages exclusively possess condensin II, such as the human pathogen *Trichomonas vaginalis*. While we identified condensin I subunits in its relative *Anaeramoeba ignava*,²² thorough sequence profile- and structure-based homology searches did not yield any candidate condensin I subunits in this species (Figure 3). Importantly, few relatives of *T. vaginalis* have been sequenced, impeding a robust comparative approach and a more precise pinpointing of the loss of condensin I.

Additionally, whereas condensin II was thought to be entirely absent in fungi,²³ we detected it in the fungus *Gonapodya prolifera* and in its sister species *Gonapodya* sp. JEL0774, confirming that the presence of condensin II was not due to contamination. Although a possibility, our protein phylogenies do not unequivocally demonstrate that these species acquired condensin II via horizontal gene transfer (HGT; data available
<figure><img src="image_8.png" /><figcaption>Figure 3. Distributions of SMC complex subunits across the eukaryotic tree of life</figcaption></figure>

Species referred to in the text are displayed in bold. Bottom: illustrations of the eukaryotic SMC complexes with their subunits. **Condensin I and II** have the SMC proteins SMC2 and SMC4, the kleisin protein CAPH (condensin I) or CAPH2 (condensin II), and the hawk proteins CAPG and CAPD2 (condensin I) or CAPG2 and CAPD3 (condensin II). The **SMC5/6** complex has the SMC proteins SMC5 and SMC6, the kleisin protein Nse4, and the kite proteins Nse1 and Nse3. It also contains Nse2, Nse5, and Nse6. **Cohesin** has the SMC proteins SMC1 and SMC3, the kleisin proteins Scc1 (mitosis) or Rec8 (meiosis), and the hawk proteins Scc3, PDS5, and/or NIPBL. Other (transient) subunits and interactors include MAU2, WAPL, Eco1, Securin, Sororin, Haspin, Shugoshin, Separase, and CTCF. See also Tables S1 and S2. Created with BioRender.

ciliate lineages.²⁵ Upon functional differ-
entiation, these expanded repertoires
likely enable ciliates to independently
orchestrate their distinct somatic and
germline genomes.

LECA harbored a complete SMC5/6
complex, including Nse5 and Nse6

Our analyses strongly suggest that
LECA harbored a complete SMC5/6
complex, including the core subunits
SMC5, SMC6, kleisin Nse4, and the kites
Nse1 and Nse3, as well as the accessory
proteins Nse2, Nse5, and Nse6. Our
expanded taxon sampling suggests that
the SMC5/6 complex was lost at least
eight times during eukaryotic evolution
(Figure 3; Table S1). These losses range
from ancient events, such as in the
ancestor of ciliates, which may have lived
over 1 Ga years ago,²⁶ to more recent
occurrences, including in the metazoan
blood fluke *Schistosoma mansoni* and
the rat parasite *Strongyloides ratti*.

Interestingly, the primary subunits
SMC5,6 and Nse1,3,4, along with the

associated SUMO ligase Nse2, consistently co-occur across
species. This co-occurrence suggests that Nse2 is integral to
the SMC5/6 complex, functioning as a core component since
before LECA, and that the complex's SUMOylation activity is
an ancient feature.

Nse5 and Nse6 were previously thought to be taxonomically
restricted and pose unique challenges for ortholog detection
owing to their rapid sequence divergence. Consequently, direct
sequence comparisons did not identify homology between the
fission yeast Nse5 and its human counterpart SLF1,²⁷ which
were considered functional analogs, not homologs. However,

at Zenodo: https://doi.org/10.5281/zenodo.15373509), leaving the exact evolutionary history of condensin II in fungi unclear. Regardless, the presence of condensin II in *Gonapodya* raises the question of the existence of chromosome territories in this genus.

Finally, we confirm that the ciliate *Tetrahymena thermophila*
expanded its condensin I repertoire in the absence of condensin
II.<sup>24,25</sup> Although the phylogenies of CAPH, CAPG, and CAPD2
proteins are not well resolved (data available at Zenodo:
https://doi.org/10.5281/zenodo.15373509), they suggest that
these proteins may have duplicated in parallel across different
using sensitive sequence searches, structure prediction, and similarity analyses, we found that Nse5 counterparts in diverse model organisms are, in fact, homologous. The same is true for Nse6 equivalents. Remarkably, we identified a putative Nse5 in *Drosophila melanogaster* (gene Dmel\CG5664), a species previously thought to lack Nse5 and Nse6.²⁸ Using structural similarity searches, we confirmed that the *D. melanogaster* Nse5 candidate strongly resembles Nse5 proteins in human, mouse, and fission yeast and is present in a range of Diptera species (STAR Methods).

Despite detecting Nse5 and Nse6 across a diverse range of eukaryotes, their distributions remain sparse in species possessing other SMC5/6 subunits. This may reflect the difficulty in identifying such highly divergent orthologs rather than true absences. This divergence is also exemplified by the additional domains found in Nse5 and Nse6 in various lineages, like ankyrin and BRCA1 C-terminal repeats in some Nse5 orthologs.²⁹

## Cohesin displays a mosaic of divergent members and partners

The presence of SMC1 and SMC3 in nearly all eukaryotes analyzed suggests a near-ubiquitous distribution of cohesin (Figure 3; Table S1). However, the distribution of non-SMC subunits is more sporadic and inconsistent, with different subunits absent in various lineages, obscuring any clear functional and evolutionary modules among these components.

Cohesin includes two kleisins, Scc1 and Rec8, functioning in mitosis and meiosis, respectively.³⁰ The wide but patchy distributions of these paralogs (Figure 3), combined with their phylogeny (STAR Methods; data available at Zenodo: https://doi.org/10.5281/zenodo.15373509), suggest that Scc1 and Rec8 resulted from a pre-LECA gene duplication and that both were present in LECA and were then independently lost multiple times. The limited sequence divergence between Scc1 and Rec8 suggests that their functions have remained closely related, with the mitosis-meiosis differentiation possibly incomplete in LECA. This, in turn, could explain the frequent loss of one of the two paralogs owing to potential functional redundancy.

The cohesin complex also contains three hawk proteins: PDS5, NIPBL, and Scc3. All three are relatively sparsely distributed, suggesting that they are also partially functionally redundant, in line with the evidence showing that PDS5 and NIPBL exclude each other from binding to the kleisin.³¹,³² Yet, various lineages have multiple paralogs of individual hawks, including humans, which have multiple copies of Scc3 (SA1/2/3) and PDS5 (PDS5A/B). However, these paralogs were derived from recent gene duplications, and we infer that LECA possessed a single copy each of PDS5, NIPBL, and Scc3. Cohesin function is modulated by several regulators, including Securin and Sororin, both crucial for sister chromatid cohesion. Our re-evaluation of Securin's characterized analogs across model organisms confirmed their homology.³³ Similarly, we confirm the homology of candidate Sororin proteins³⁴ and their broad distribution. Both of these largely disordered regulators were detected primarily through the presence of short linear motifs, which are not always unique, raising the possibility of convergent evolution in some cases (STAR Methods). Altogether, the broad presence of Se-

curin and Sororin across eukaryotes suggests that these proteins were already regulating cohesin in LECA.

## Eukaryotes inherited an expanded SMC array from archaeal ancestors

### SMC proteins

To investigate the pre-LECA origins of the eukaryotic SMC complexes, we performed phylogenetic analyses. These confirm that eukaryotic SMC proteins form two clusters,¹⁰,³⁵ one consisting of condensin and cohesin SMC proteins and the other of SMC5 and SMC6, separated by several clades of prokaryotic homologs (Figure 4A). However, topology tests suggest that the possibility that these two clusters actually form a monophyletic group cannot be rejected (*p*-approximately unbiased [AU] = 0.317). Both condensin/cohesin SMC proteins and SMC5,6 show archaeal homologs as their closest relatives, including Asgard archaea, indicating that eukaryotes inherited them from their archaeal ancestors.

Several archaea, particularly Thaumarchaeota-Aigarchaeota-Crenarchaeota-Korarchaeota (TACK) and Asgard archaea, have two SMC proteins (Table S3), likely resulting from a gene duplication event in an early archaeal ancestor. Our analyses confirm that after eukaryotes diverged from their archaeal ancestors, the ancestral condensin/cohesin SMC protein underwent an additional duplication event, resulting in the ancestors of SMC1,4 and SMC2,3. Similarly, the ancestor of SMC5,6 duplicated to give rise to SMC5 and SMC6.¹⁰ In both cases, the duplication resulted in a distinct κ-SMC (Figure 2A, SMC1,4, and 5) and ν-SMC (SMC2,3, and 6), while in prokaryotes, these interacting partners are the same protein. Thus, these duplications and subfunctionalizations within the SMC protein complexes occurred in parallel, yielding SMC protein heterodimers in eukaryotic SMC complexes rather than the homodimers seen in prokaryotes. After this heterodimer-forming duplication, the condensin/cohesin SMC proteins duplicated once more, giving rise to the condensin and cohesin SMC protein heterodimers.

### Kleisin proteins

In contrast to previous work,¹⁰ our phylogeny suggests that the kleisins from condensin/cohesin and from SMC5/6 do not form a monophyletic group (Figure 4B). Their monophyly was rejected by the AU topology test (*p*-AU = 0.002). Interestingly, the SMC5/6 kleisin, Nse4, branches with strong support together with a clade containing kleisins from TACK and Asgard archaea, suggesting an Asgard archaeal provenance of Nse4, in line with the Asgard archaeal ancestry of eukaryotes.¹⁴,¹⁵ By contrast, while we observe other clades containing a wide representation of archaea, they do not form a monophyletic group with condensin/cohesin kleisins.

We hypothesize that these non-Nse4-related archaeal clades, which contain the same organismal diversity as the Nse4-related archaeal clade (i.e., TACK and Asgard archaea), represent the true sister group of the condensin/cohesin kleisins. This is supported by inspection of the genomic context of this group of archaeal kleisins, which shows them encoded next to condensin/cohesin-related SMC proteins (Figure 4D; STAR Methods). The duplication of kleisin in an archaeal ancestor would be coherent with the duplication scenario for the SMC proteins outlined above. Within the condensin/cohesin kleisin clade, it is not
<figure><img src="image_9.png" /><figcaption>**Figure 4. Phylogenetic relationships between eukaryotic and prokaryotic SMC complexes**</figcaption></figure>

(A–C) Phylogenetic trees of SMC (A), kleisin (B), and kite (C) proteins, inferred using IQ-TREE³⁶ under the LG+C60+G mixture model. Statistical support at branches was estimated from 1,000 ultrafast bootstrap replicates.³⁷ Uncollapsed, unfiltered trees can be found on https://itol.embl.de/tree/13722425212266531697468040 (SMC), https://itol.embl.de/tree/137224252126461697465203 (kleisin), and https://itol.embl.de/tree/13722425212198711697525255 (kite). Scale bars indicate estimated average numbers of substitutions per site.

(D) Genomic localizations of SMC complex operons, with genes encoding SMC proteins, kleisins, and kites in a selection of archaea having at least two paralogs of each. The NCBI GenBank/RefSeq genome assembly identifiers are indicated, as well as the scaffolds containing the operons. Candidatus Heimdallarchaeota archaeon LC_3 appears to have undergone a duplication of its (ScpB-like) kite subunit in its condensin/cohesin-related SMC complex operon. See also Figure S1 and Tables S2 and S3.

clear which kleisins are most closely related to one another, since these branches are poorly supported. Given the putative close relationship of condensin I and II, which share SMC proteins, we hypothesize that the condensin kleisins CAPH and CAPH2 are more closely related to each other than to the cohesin kleisins, a hypothesis also hinted at by a more restricted phylogenetic analysis (STAR Methods; Figure S1).

In summary, eukaryotic SMC proteins and kleisins appear to have originated from an ancient archaeal gene duplication, probably in the ancestor of TACK and Asgard archaea, which was followed by further duplications specific to eukaryotic evolution.

## Kite proteins

Our identification of prokaryotic kite homologs of Nse1 and Nse3 (SMC5/6 complex) suggests that, at the sequence level, prokaryotic kites fall into three distinct archetypes: Nse1/3-like (archaeal kites only), ScpB-like (both archaeal and bacterial kites, such as ScpB in *Bacillus subtilis*), and MukE-like (other bacterial kites, like MukE in *Escherichia coli*). Despite their marked sequence divergence, these archetypes share similar structural features.³⁸ Our kite phylogeny mirrors that inferred from SMC and kleisin proteins, where various archaea, in particular TACK and Asgard archaea, possess two kites: one
<figure><img src="image_10.png" /></figure>

<figure><img src="image_11.png" /><figcaption>Figure 5. HMM profile network analysis and 3D protein structure comparisons of hawk proteins</figcaption></figure>

(A) Graph of a profile-versus-profile-based network analysis. The initial query profiles were those of hawk proteins, WAPL and MAU2, which were searched against the COG/KOG database of orthologous groups of proteins. All profile hits identified in the initial search were subsequently taken as query, creating a

(legend continued on next page)
Nse1/3-like kite and one ScpB-like kite (Figure 4C). Thus, the kite protein likely duplicated in the ancestor of TACK and Asgard archaea.

Our analyses of SMC protein, kleisin, and kite paralog distribu-
tions indicate that many archaea harbor two complete SMC
complexes. Further examination of their genomic localizations
in representative species reveals that the paralogs are organized
into distinct operons, each encoding all three subunits
(Figure 4D). Based on our phylogenetic inferences, we identify
these as condensin/cohesin-related and SMC5/6-related com-
plexes. Although most species possessing both complexes
belong to the TACK and Asgard archaea, a few euryarchaeal
species also carry them, in particular in Hadesarchaeota
(Table S3). As Euryarchaeota diverged from TACK and Asgard
archaea even earlier (Figure 6), this might indicate an even older
origin of the two distinct complexes. However, as Hadesarch-
aeota are uncertain to actually belong to Euryarchaeota,³⁹ and
as very few Euryarchaeota have the two complexes, we propose
that the duplication of the entire SMC complex operon occurred
in the common ancestor of TACK and Asgard archaea. The pres-
ences of the two complexes outside of TACK and Asgard
archaea might be due to rare HGT events.

Of note, some Asgard archaea possess even more kite paral-
ogs (including Hodarchaeales¹⁵), with one of them forming an
exclusively Asgard archaeal clade clustering with (eukaryotic)
Nse3 (Figure 4D). This suggests that the duplication event lead-
ing to Nse1 and Nse3 occurred in an Asgard archaeal ancestor.

Hawk and other alpha-solenoid fold proteins

Instead of kite proteins, condensin and cohesin have hawk
proteins (Figure 1). Hawk proteins are HEAT repeat proteins.
HEAT repeats comprise an alpha-solenoid fold, similar to arma-
dillo (ARM) and tetratricopeptide repeat (TPR) domains. We em-
ployed extensive homology searches to investigate the deep
evolutionary origins of the hawk proteins, yet were not able to
detect them in prokaryotes. In addition, their degenerate nature
renders their sequences too divergent to conduct conventional
phylogenetic analysis. To investigate their relationships to each
other and to other alpha-solenoids, we adopted a sequence
profile network approach⁵ combined with comparisons of
AlphaFold2-predicted 3D structures. Interestingly, our analysis
leads us, among others, to conclude that the cohesin release
factor WAPL is not a HEAT repeat protein, as previously
thought,⁴⁰,⁴¹ but an ARM repeat protein (Figure 5A; Table S4;
STAR Methods). Hence, even though hawk proteins and WAPL
both have roles in cohesin function and regulation, they do not
seem to share a relatively recent common origin. Instead,
hawk proteins seem to be more closely related to adaptins and
PP2A regulation proteins, which also harbor HEAT-type alpha-
solenoids.

Furthermore, our analysis confirms that the hawks are most
closely related to one another,⁵ indicating that they are

paralogs resulting from gene duplication events that occurred
before LECA. Interestingly, they form two groups in the profile
and structure analyses, which correspond to the previously
described functional groups in the condensin/cohesin com-
plexes⁴⁴: the A-type hawks (CAPG, CAPG2, and Scc3) and the
B-type hawks (CAPD2, CAPD3, NIPBL, and PDS5) (Figures 5A
and 5B; Tables S5, S6, and S7). A likely scenario is that, in the
ancestor of condensin and cohesin, a single hawk protein first
replaced kite(s). This protein initially formed a homodimer and
then duplicated, resulting in an A and B type-containing
heterodimer.

In addition to hawk proteins, WAPL, the cohesin loading factor MAU2, and the SMC5/6 subunits Nse5 and Nse6 also adopt an alpha-solenoid structure. This suggests that they might be homologous to hawk proteins, possibly adopting a HEAT repeat-like fold.⁴⁵,⁴⁶ We applied sequence- and structure-based analyses but were not able to classify Nse5 and Nse6 as any particular alpha-solenoid with certainty, leading to their exclusion from our network-based analysis. However, despite their extensive divergence relative to other alpha-solenoids, Nse5 and Nse6 show remarkable similarities to each other at the structural level, which we considered suggestive of homology between these proteins. To further investigate this possibility, we predicted the 3D structures of Nse5 and Nse6 orthologs and aligned them. This revealed an on-average significant similarity between predicted Nse5 and Nse6 structures from a broad sample of eukaryotic lineages (average TM-score, 0.51; Figures S2A and S2B),⁴⁷ with the best alignment being highly significant (TM-score, 0.68; Figure S2C). In the absence of other discernible closer homologs, we propose that Nse5 and Nse6 are sister paralogs resulting from a single ancestral protein that duplicated before LECA. Hence, the Nse5 and Nse6 subcomplex probably first consisted of a homodimer formed by this ancestral protein, while, after gene duplication, it was converted into the heterodimer.

DISCUSSION

An evolutionary scenario for the emergence of
eukaryotic SMC complexes

Our phylogenetic analyses of SMC proteins, kleisins, and kites
suggest an ancient duplication of the entire SMC complex in
an archaeal ancestor (Figure 6). We propose that the duplicated
SMC proteins formed separate homodimers rather than a single
heterodimeric complex, which is supported by the genomic co-
localization of each complex's candidate subunits in extant
archaeal genomes (Figure 4D).

The SMC complex duplication event likely occurred in the
ancestor of TACK and Asgard archaea. We propose that,
during the origination of eukaryotes (eukaryogenesis), each
complex underwent SMC protein duplication and subsequent

<footnote>bidirectional graph. Displayed edges are filtered to show only significant hits (E value ≤ 1E−3, alignment length ≥ 100, which corresponds to ~2 HEAT repeat units). Nodes are colored based on MCL cluster identity. See also Tables S5 and S6.

(B) Predicted structures of hawk proteins of *Homo sapiens* (*Hs*) and *Arabidopsis thaliana* (*At*) (STAR Methods). Pairwise alignment Z scores as computed by Dali⁴² are represented in the heatmap, generated with ClustVis.⁴³ The estimated accuracies of the protein structure predictions are represented by predicted local distance difference test (pLDDT) scores.

See also Table S7.</footnote>
<figure><img src="image_12.png" /></figure>

<caption>Figure 6. Evolutionary scenario for the origins of the four eukaryotic SMC complexes between the FECA and LECA, referred to as eukaryogenesis</caption>

The first eukaryotic common ancestor (FECA) possessed two distinct SMC complexes, a legacy of an early archaeal duplication event of the SMC complex, which we hypothesize to have occurred in the ancestor of TACK and Asgard archaea (the leftmost black star). One of these complexes underwent a duplication of both its SMC and kite proteins, leading to the evolution of the SMC5/6 complex. Note that the timing of the kite duplication is unresolved; it might either have occurred before FECA (it would be shared with Asgard archaea; Figure 4C) or after FECA. The SMC protein duplication transformed the original SMC protein homodimer within the complex into a heterodimer, comprising a κ-SMC and a ν-SMC.

The second complex also underwent a duplication

of its SMC protein. However, in this instance, the kite subunits were replaced by hawk proteins. This SMC complex underwent an additional duplication, resulting in the emergence of cohesin and condensin. Subsequently, the condensin complex underwent a partial duplication, specifically affecting its non-SMC proteins: kleisin and hawks. Finally, within the ancestral condensin/cohesin, the hawk proteins also duplicated. The timing of the hawk duplications is speculative, as we were not able to conduct a phylogenetic analysis for this family. The composition of SMC complexes is illustrated in Figures 2A and 3. Created with BioRender.

subfunctionalization, generating heterodimers composed of κ-SMC and ν-SMC subunits (Figure 6). The proposed SMC protein duplication occurred in parallel in both the condensin/cohesin ancestor as well as in the SMC5/6 complex ancestor. Indeed, such homomer-to-heteromer duplications are very common in eukaryotic evolution,⁴⁸,⁴⁹ yet they might not necessarily reflect any selective advantage.⁵⁰,⁵¹ In the condensin/cohesin ancestor, the ScpB-like kite protein was replaced by a hawk protein. This coincided with kleisin elongation, as condensin and cohesin kleisins are longer than those in the SMC5/6 complex and prokaryotic SMC complexes.⁵,⁴⁴ While phylogenetic analyses of hawk proteins were hampered by their extensive divergence, our network analysis suggests that the first hawk formed a heterodimer, which likely duplicated and formed a heterodimer of A- and B-type hawks.⁴⁴ This complex then duplicated, giving rise to condensin and cohesin. Condensin experienced a partial duplication, affecting only the kleisin and hawks, resulting in condensin I and II. Additionally, cohesin's kleisin and one of its hawks duplicated. In the SMC5/6 ancestor, the kite protein duplicated, likely before the divergence of eukaryotes from Asgard archaea (Figure 4C).

Beyond these duplication events, the SMC5/6 complex and cohesin recruited additional subunits and regulators (e.g., Nse2 and Nse5/6 for SMC5/6 and WAPL, Securin, and Sororin for cohesin). Existing members also acquired new domains; for example, Nse1 gained a RING-finger E3 ubiquitin ligase domain,⁵² absent in Nse3 and archaeal kites. This eukaryotic innovation may enable SMC5/6 to ubiquitinate proteins,⁵³ just as the addition of Nse2 enabled it to SUMOylate proteins. Combined, our data demonstrate that evolutionary mechanisms, including gene duplication, replacement, and recruitment, endowed LECA with the four SMC complexes found in modern eukaryotes. Our analysis of SMC complex evolution highlights the importance of gene duplication in eukaryotic complexity,⁵⁴,⁵⁵ as it involved at least two whole-complex duplications, one partial-complex duplication, and multiple intra-complex duplications.

## Outlook: Toward evolutionary cell biology of SMC complexes

The distributions of SMC complexes across eukaryotes raise questions about their functional differences. For example, what roles does condensin I have in species lacking condensin II? Do condensin complexes fulfill any non-mitotic functions, such as forming TADs⁵⁶,⁵⁷? Do species lacking condensin II exhibit a Rab-like nuclear architecture rather than a chromosome territory one? Several of the species in our dataset do not conform to the latter correlation. For example, *Plasmodium falciparum* and *Dictyostelium discoideum* possess condensin II and yet have a Rab-like organization,⁵⁸,⁵⁹ while the moss *Physcomitrium patens* lacks condensin II and forms chromosome territories.⁶⁰ Scrutinizing this relationship experimentally would be particularly relevant in fungi, where we identified condensin II in the chytrid lineage of *Gonapodya*. Furthermore, exploring lineages where SMC5/6 has been recently lost, like in the nematode *S. ratti*, could enhance our understanding of the dispensability of this complex.

In addition to raising questions about the implications of protein presences and absences, our ortholog sets allow for an in-depth examination of individual protein sequences, which may reveal new sequence motifs mediating functions, for example via protein-protein interactions. As our work comprises a wide range of SMC complex proteins, many different researchers might profit from this possibility. It is important to point out that what constitutes an SMC complex protein is somewhat blurred, with some proteins being only loosely associated or transient interactors, which we included in order to offer a comprehensive overview.

The relationship between nuclear chromosome architecture and condensin II remains to be confirmed across a wider, more diverse range of species. If true, it would pave the way to address questions about the cellular consequences of the frequent losses of the condensin II complex. In particular, transitioning from chromosome territories to a Rab-like architecture

<footnote>*Hodarchaeales: order within Asgard archaea, closest identified prokaryotic relatives of eukaryotes*¹⁵</footnote>
as a result of loss of condensin II, may lead to decreased chromatin flexibility owing to chromosome tethering to the nuclear envelope⁶¹ and therefore reduce precise and context-dependent control of gene expression. Yet, the functional role of chromosome territories has been questioned.⁶² At the same time, a Rabl-like configuration might allow cells to accelerate their cell cycle, as it requires less reorganization before going into mitosis.⁶³ Such a configuration could be especially advantageous in life histories involving rapid cell divisions, as seen in certain parasitic or rapidly growing fungi.

Future investigations comparing closely related species or
strains that differ in condensin II presence would help dissect
whether (simpler) nuclear organization correlates with faster
cell cycles and possibly diminished regulatory plasticity. Such
studies could use imaging or Hi-C-based approaches to explore
how nuclear architecture dynamics influence gene expression,
replication timing, and genomic stability. Ultimately, clarifying
the balance between these potential benefits (e.g., faster proliferation) and costs (e.g., reduced chromatin flexibility) could illustrate how chromosome configuration evolves in diverse eukaryotic lineages.

Our findings indicate that diverse archaea, particularly within
the TACK and Asgard groups, harbor both condensin/cohesin-
related and SMC5/6-related complexes. This hypothesis should
be tested through experimental investigation of extant archaea
to confirm the presence and functionality of these two distinct
SMC complexes in vivo. Although challenging, recent culturing
successes of Asgard archaeal lineages⁶⁴⁻⁶⁶ might allow for
such studies; for example, in *Prometheoarchaeum syntrophicum*
(Figure 4D). These promise to validate our predictions while also
offering a unique opportunity to elucidate the ancestral functions
of condensin/cohesin and SMC5/6. The characterization of the
SMC5/6-related complex in archaea is of particular interest,
given the ongoing uncertainty regarding its canonical eukaryotic
function.

Altogether, the evolutionary trajectories of SMC complexes
provide a starting point for studying the genetic underpinnings
of eukaryotic genome organization (Figure 1). In particular, they
might help to understand how and why eukaryotes differ in their
genome organization and how their organizational complexity
emerged during eukaryogenesis.⁶⁷

Limitations of the study

Our evolutionary analysis of SMC complexes provides signifi-
cant insights into their origins and distribution, but several limita-
tions warrant consideration.

First, despite our extensive dataset of 177 eukaryotic and
4,101 prokaryotic proteomes, sequencing biases toward model
organisms and certain taxonomic groups may obscure the full
picture of SMC complex evolution. This is particularly relevant
for free-living microbial eukaryotic lineages that remain under-
sampled, potentially concealing certain evolutionary events or
preventing us from more precisely timing them.

Second, the highly divergent nature of certain SMC complex
proteins, such as Nse5 and Nse6, presents challenges for ho-
mology detection. Despite employing sensitive sequence profile
searches and structural predictions and comparisons, we
cannot exclude the possibility of false negatives, especially in

rapidly evolving lineages. This limitation is also relevant for disor-
dered proteins like Securin and Sororin, where short linear motifs
guided our homology assessment, while they might, in certain
cases, be misleading due to their potential convergent emer-
gence in non-homologous proteins (STAR Methods).

Third, incomplete or fragmented genome assemblies may
lead to an apparent absence of SMC complex components
that are actually present but not detected (see Table S1 for
BUSCO⁶⁸ completeness estimates). We attempted to mitigate
this by being stringent in inferring the loss of an entire complex,
for which we required all complex-specific subunits to be ab-
sent. Still, genome quality remains a potential confounding factor
in our distribution analysis.

Fourth, while our phylogenetic analyses support the origina-
tion of both a condensin/cohesin-related complex and an
SMC5/6-related complex in the ancestor of TACK and Asgard
archaea, deep branches in the trees often lack strong statistical
support, and alternative topologies cannot be definitively ruled
out. Moreover, we noticed the extremely rare presence of these
two complexes in Euryarchaeota (Table S3), which might point to
an even older origin of these two complexes if this presence does
not result from a different topology of the archaeal tree of life,
HGT events, or data contamination. Furthermore, the extensive
sequence divergence of hawk proteins prevented conventional
phylogenetic analysis, limiting our understanding of their precise
evolutionary relationships.

Finally, our genomics-based approach cannot directly infer
the functional implications of SMC complex composition. While
we propose correlations between condensin II absence and
Rabl-like nuclear architecture based on previous literature,¹²
functional validation across diverse eukaryotes is needed to
confirm these relationships. Similarly, our inferences about
archaeal SMC complexes remain speculative without experi-
mental validation in extant archaea.

RESOURCE AVAILABILITY

Lead contact

Requests for further information and data should be directed to and will be ful-
filled by the lead contact, Jolien J.E. van Hooff (jolien.vanhooff@wur.nl).

Materials availability

No materials were generated in this study.

Data and code availability

* Original code and additional data have been deposited at Zenodo at https://doi.org/10.5281/zenodo.15373509 and are publicly available as of the date of publication.

* Any additional information required to reanalyze the data reported in this paper is available from the [lead contact](#) upon request.

ACKNOWLEDGMENTS

We kindly thank Miguel Andrade (Johannes Gutenberg University) for helpful
advice and expert assessment of the classification of the alpha-solenoid folds
of Nse5 and Nse6; Jason Stajich (University of California, Riverside) for sharing
the predicted protein sequences of *Gonapodya* sp. JEL0774; Bastiaan de
Potter, Laura van Rooijen, Carlos Santana-Molina, and Berend Snel (Utrecht
University) for collaborating in compiling the eukaryotic proteome dataset;
and Benjamin Rowland (Netherlands Cancer Institute), Chris Lane (University
of Rhode Island), and Michelle Leger (Okinawa Institute of Science and
Technology) for constructive feedback on an earlier version of this manuscript. This work was supported by the European Research Council (ERC Starting Grant 2018, grant 803151, to L.E.) and the Dutch Research Council (NWO, grant VI.Veni.212.099 to J.J.E.v.H. and grant VI.Veni.202.223 to E.C.T.).

## AUTHOR CONTRIBUTIONS

Conceptualization, J.J.E.v.H., E.C.T., and L.E.; data curation, J.J.E.v.H., M.W.D.R., and E.C.T.; analysis, J.J.E.v.H., M.W.D.R., and E.C.T.; methodology, J.J.E.v.H., M.W.D.R., E.C.T., and L.E.; project administration, J.J.E.v.H.; resources, J.J.E.v.H., E.C.T., and L.E.; supervision, J.J.E.v.H., E.C.T., and L.E.; validation, J.J.E.v.H., M.W.D.R., and E.C.T.; visualization, J.J.E.v.H., M.W.D.R., and E.C.T.; writing—original draft, J.J.E.v.H. and L.E.; writing—review and editing; J.J.E.v.H., M.W.D.R., E.C.T., and L.E.

## DECLARATION OF INTERESTS

The authors declare no competing interests.

## DECLARATION OF GENERATIVE AI AND AI-ASSISTED TECHNOLOGIES

During the preparation of this work, the authors used ChatGPT, Claude, and Gemini in order to improve the clarity of the writing. After using these tools, the authors reviewed and edited the content as needed and take full responsibility for the content of the publication.

## STAR★METHODS

Detailed methods are provided in the online version of this paper and include the following:

* KEY RESOURCES TABLE

* METHOD DETAILS

* Eukaryotic dataset assembly

* Ortholog detection in eukaryotes

* Phylogenetic and network analysis in eukaryotes and prokaryotes

* Visualization

* QUANTIFICATION AND STATISTICAL ANALYSIS

## SUPPLEMENTAL INFORMATION

Supplemental information can be found online at https://doi.org/10.1016/j.celrep.2025.115855.

Received: December 24, 2023

Revised: April 8, 2025

Accepted: May 27, 2025

Published: June 19, 2025

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## STAR★METHODS

### KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td colspan="3"><strong>Deposited data</strong></td></tr><tr><td>profile HMMs, full and trimmed alignments, phylogenetic trees, overviews of prokaryotic and eukaryotic taxa, scripts for collecting phyletic patterns and for generating Figure 3 in iTOL</td><td>GitHub</td><td>https://github.com/jolienvanhooff/SMCevolution<br>Published release at Zenodo: https://doi.org/10.5281/zenodo.15373509</td></tr><tr><td>Full phylogenetic trees for SMC, kite and kleisin proteins (related to Figure 4)</td><td>iTOL</td><td>SMC proteins: https://itol.embl.de/tree/13722425212266531697468040<br>kleisin family: https://itol.embl.de/tree/137224252126461697465203<br>kite family: https://itol.embl.de/tree/13722425212198711697525255</td></tr><tr><td colspan="3"><strong>Software and algorithms</strong></td></tr><tr><td>BUSCO v5.2.2</td><td>Manni et al.<sup>68</sup></td><td>RRID:SCR_015008; http://busco.ezlab.org/</td></tr><tr><td>BLAST v2.13.0+</td><td>Altschul et al.<sup>69,70</sup></td><td>RRID:SCR_004870; https://blast.ncbi.nlm.nih.gov/Blast.cgi</td></tr><tr><td>MAFFT v7.515</td><td>Katoh and Standley<sup>71</sup></td><td>RRID:SCR_011811; https://mafft.cbrc.jp/alignment/software/</td></tr><tr><td>MAFFT-DASH v7.515</td><td>Rozewicki et al.<sup>72</sup></td><td>https://sysimm.org/dash/</td></tr><tr><td>BMGE v1.12</td><td>Criscuolo and Gribaldo<sup>73</sup></td><td>https://gensoft.pasteur.fr/docs/BMGE/1.12/BMGE_doc.pdf</td></tr><tr><td>IQ-TREE v2.0.3</td><td>Minh et al.<sup>36</sup></td><td>RRID:SCR_017254; http://www.iqtree.org/</td></tr><tr><td>PANTHER17.0</td><td>Thomas et al.<sup>74</sup></td><td>RRID:SCR_004869; http://www.pantherdb.org/</td></tr><tr><td>EggNOG v6.0</td><td>Hernández-Plaza<sup>75</sup></td><td>RRID:SCR_002456; http://eggnog.embl.de</td></tr><tr><td>HMMER v.3.3.2</td><td>Eddy<sup>76</sup></td><td>RRID:SCR_005305; http://hmmer.org</td></tr><tr><td>HMMER-jackhmmer (web server)</td><td>Potter et al.<sup>77</sup></td><td>https://www.ebi.ac.uk/Tools/hmmer/(this website has been phased out)</td></tr><tr><td>MPI Bioinformatics Toolkit-HHpred (web server)</td><td>Zimmerman et al.<sup>78</sup></td><td>RRID:SCR_010276; https://toolkit.tuebingen.mpg.de/tools/hhpred</td></tr><tr><td>AFDB</td><td>Varadi et al.<sup>79</sup></td><td>https://alphafold.ebi.ac.uk/</td></tr><tr><td>Dali (web server)</td><td>Holm<sup>42</sup></td><td>RRID:SCR_013433; http://ekhidna.biocenter.helsinki.fi/dali_server</td></tr><tr><td>Foldseek (web server)</td><td>van Kempen et al.<sup>80</sup></td><td>https://search.foldseek.com/search</td></tr><tr><td>GTDB r207</td><td>Parks et al.<sup>81</sup></td><td>https://gtdb.ecogenomic.org/</td></tr><tr><td>CheckM</td><td>Parks et al.<sup>82</sup></td><td>RRID:SCR_016646; http://ecogenomics.github.io/CheckM/</td></tr><tr><td>CD-HIT v4.8.1</td><td>Fu et al.<sup>83</sup></td><td>RRID:SCR_007105;</td></tr><tr><td>trimAl v1.4.1</td><td>Capella-Gutiérrez et al.<sup>84</sup></td><td>RRID:SCR_017334; http://trimal.cgenomics.org/</td></tr><tr><td>PROMALS3D (web server)</td><td>Pei and Grishin<sup>85</sup></td><td>RRID:SCR_018161; http://prodata.swmed.edu/promals3d/promals3d.php</td></tr><tr><td>UFBOOT2</td><td>Hoang et al.<sup>37</sup></td><td>RRID:SCR_017254; http://www.iqtree.org/</td></tr><tr><td>HH-suite v3.3.0</td><td>Steinegger et al.<sup>86</sup></td><td>RRID:SCR_016133; https://github.com/soedinglab/hh-suite</td></tr><tr><td>clusterMaker2</td><td>Morris et al.<sup>87</sup></td><td>https://www.rbvi.ucsf.edu/cytoscape/clusterMaker2/</td></tr><tr><td>Cytoscape v3.9.1</td><td>Shannon et al.<sup>88</sup></td><td>RRID:SCR_003032; http://cytoscape.org</td></tr></tbody></table>

(Continued on next page)
<figure><img src="image_13.png" /></figure>

**Continued**

<table><thead><tr><td>REAGENT or RESOURCE</td><td>SOURCE</td><td>IDENTIFIER</td></tr></thead><tbody><tr><td>TM-align v.20190822</td><td>Zhang and Skolnick<sup>89</sup></td><td>RRID:SCR_024390; https://zhanglab.ccmb.med.umich.edu/TM-align/</td></tr><tr><td>PyMOL</td><td>Schrödinger<sup>90</sup></td><td>RRID:SCR_000305; http://www.pymol.org/</td></tr><tr><td>ITOL</td><td>Letunic and Bork<sup>91</sup></td><td>RRID:SCR_018174; https://itol.embl.de/</td></tr><tr><td>UCSF ChimeraX v1.8</td><td>Meng et al.<sup>92</sup></td><td>RRID:SCR_015872; https://www.cgl.ucsf.edu/chimerax/</td></tr><tr><td>ETE v3</td><td>Huerta-Cepas et al.<sup>93</sup></td><td>https://etetoolkit.org/</td></tr><tr><td>BioPython v1.79</td><td>Cock et al.<sup>94</sup></td><td>RRID:SCR_007173; https://biopython.org/</td></tr><tr><td>IntAct</td><td>Orchard et al.<sup>95</sup></td><td>RRID:SCR_006944; http://www.ebi.ac.uk/intact</td></tr><tr><td>AlphaFold2</td><td>Jumper et al.<sup>96</sup></td><td>RRID:SCR_025454; https://github.com/google-deepmind/alphafold</td></tr><tr><td>ColabFold</td><td>Mirdita et al.<sup>97</sup></td><td>RRID:SCR_025453; https://github.com/sokrypton/ColabFold</td></tr><tr><td>pdb-tools</td><td>Rodrigues et al.<sup>98</sup></td><td>https://www.bonvinlab.org/pdb-tools/</td></tr><tr><td>ggplot2</td><td>Wickham<sup>99</sup></td><td>RRID:SCR_014601; https://cran.r-project.org/web/packages/ggplot2/index.html</td></tr><tr><td>Jalview</td><td>Waterhouse et al.<sup>100</sup></td><td>RRID:SCR_006459; http://www.jalview.org/</td></tr></tbody></table>

## METHOD DETAILS

### Eukaryotic dataset assembly

To determine the presences and absences of eukaryotic SMC complex subunits across the eukaryotic Tree of Life, and to reconstruct their evolutionary histories, we used a eukaryotic dataset that we compiled previously.¹⁰¹ This dataset contains the protein sequences of phylogenetically diverse eukaryotes, and model organisms. For this project, we modified the dataset as described before.¹⁰² An overview of the included species can be found in Table S1, where we also report the proteome completeness scores derived from BUSCO.⁶⁸ While we attempted to remove redundancy from protein sets, some sets still contain some highly similar sequences, very likely representing isoforms encoded by the same gene. Therefore, some apparent species-specific paralogs in the identified orthologs for a given protein might not correspond to different loci, but to a single locus.

### Ortholog detection in eukaryotes

#### Protein selection and ortholog definition

As the subjects of our study, we selected the subunits of condensin I, condensin II, cohesin and SMC5/6. Additionally, we selected proteins regulating these complexes, provided that they are specific (i.e., no general cell cycle regulators like PP2A, CDK1, and Aurora kinases). The selected proteins should have been characterized as a complex component in at least one organism, predominantly *H. sapiens*, *S. cerevisiae*, and *S. pombe*,²⁸,¹⁰³ and also *T. thermophila*,²⁴,²⁵,¹⁰⁴,¹⁰⁵ *P. patens*,¹⁰⁶,¹⁰⁷ *A. thaliana*,³⁴,¹⁰⁸ and *C. merolae*.¹⁰⁹

For each selected protein, our objective was to detect its orthologs among the protein sequences of the species in our eukaryotic dataset. Here, 'orthologs' are defined as descendants from a single ancestral protein in LECA, or in a more recent lineage, if the protein is taxonomically restricted to a particular eukaryotic clade. As a result, groups of orthologs include paralogs resulting from recent duplications, that is, more recent than LECA. For example, PDS5A,B are both members of the group of PDS5 orthologs.

#### Ortholog calling: Two global approaches

Each protein required its own, tailored approach to ortholog calling, of which the details can be found in Table S2 (sheet 'Orthologs').

Our approaches can be globally described by two different strategies. First, for the SMC proteins SMC1-6, we searched for homologs in our eukaryotic dataset with BLASTP⁶⁹ (E-value cutoff 1e-5) using previously identified SMC protein sequences.¹⁰ We combined SMC protein sequences from¹⁰ with our hits and aligned all of them with MAFFT (v7, settings: -auto),⁷¹ trimmed the alignment with BMGE (v1.12, settings: -m BLOSUM30, -b 3 -h 0.7 -g 0.7)⁷³ and inferred an initial maximum likelihood phylogeny using IQ-TREE (v2.0.3, settings: -m LG + F + R5 -ar1t 1000).³⁶ From this phylogeny, we extracted the sequence belonging to each of SMC1-6, for which we subsequently made protein-specific profile HMMs. We used these HMMs to locally run hmmsearch (hmmer.org) across our eukaryotic dataset. We gathered the hit sequences using a domain bitscore cut-off that was well below the lowest-scoring members of putative orthologs in the initial phylogeny, to ensure all orthologs were included. For these hit sequences, we inferred a final phylogeny from which the orthologs were extracted.
<figure><img src="image_14.png" /></figure>

Second, for non-SMC proteins, we derived profile HMMs from PANTHER17.0<sup>74</sup> and EggNOG v6.0.<sup>75</sup> We used these HMMs to search across our eukaryotic dataset, and verified that characterized orthologs and/or functional analogs were hit by it. Then, we performed at least one iteration of the following steps: construct a profile HMM of the hits and search across our eukaryotic dataset, verify the new hits manually, and gather the trusted hits. In the next iteration, the trusted hits served as input for a new profile HMM. As long as new hits were found, we continued this cycle. For highly divergent proteins, we adopted additional strategies. These involved sequence-based strategies, like BLASTP/PSI-BLAST,<sup>69,70</sup> online jackhmmer<sup>77</sup> and online HHpred,<sup>110</sup> frequently using multiple query sequences. Since sequences typically are less conserved than structures, we also adopted structure-based strategies, which entail searching for similar structures using experimentally determined structures or predicted structures from the AlphaFold Protein Structure Database,<sup>79</sup> applying search algorithms Dali<sup>42</sup> and Foldseek,<sup>80</sup> also often using multiple query structures. The collected homologs were aligned to infer a maximum likelihood phylogeny (details in Table S2, sheet 'Orthologs'), from which the orthologs were extracted. If, because of strong sequence divergence, we expected the orthologous cluster in the phylogeny to be incomplete, we used the orthologs in the cluster to build a profile HMM to again search through our dataset, infer another phylogeny and extract the orthologs, potentially expanded compared to the previous set.

For all orthologous groups, we manually checked conspicuous absences and putative highly divergent orthologs using hmmsearch -max (HMM searches without heuristic filters),<sup>76</sup> FoldSeek<sup>80</sup> (using AF2/3-predicted protein structures<sup>90</sup>) and/or HHpred.<sup>78</sup> Relevant information about these searches can be found in our online repository (https://github.com/jolienvanhooff/SMCevolution, published release at Zenodo: https://doi.org/10.5281/zenodo.15373509).

We verified the absence of the condensin II complex from the model plant *P. patens* by searching for orthologs of CAPH2, CAPG2 and CAPD3 in the recently published near telomere-to-telomere assembly of this species,<sup>60</sup> which was not yet in our dataset. Indeed, we could not find candidate orthologs of these condensin II proteins in the predicted proteins of this new assembly.

We confirmed our *D. melanogaster* Nse5 candidate by first searching for its homologs with BLASTP versus NR (max. 250 hits, a permissive E-value cutoff of 1). This yielded 122 hits among a wide range of Diptera, including for instance the mosquito *Aedes aegypti* (gene 5579981, E-value = 3e-09). We confirmed both the *D. melanogaster* and the *A. aegypti* Nse5 candidates to be structurally similar to Nse5 proteins in human, mouse and fission yeast using Foldseek<sup>80</sup> versus AlphaFold/Swiss-Prot v4. Of note, the IntAct<sup>95</sup>-reported interactors of the *D. melanogaster* Nse5 candidate seem to fulfill neural functions, and include no apparent Nse6 candidate. Thus far we have failed to detect a *D. melanogaster* Nse6 homolog.

### A tailored, motif-based approach to homolog calling for Securin and Sororin

Cohesin regulators Securin and Sororin are predominantly composed of intrinsically disordered regions, which accounts for their low conservation across eukaryotes. Consequently, for identifying their orthologs, we have primarily relied on short protein motifs often associated with protein-protein interactions. However, the mere presence of such a motif does not necessarily designate a protein as an ortholog, as these simple motifs can arise through convergent evolution.

Securin has been described in human and yeast, and a candidate has been identified in *Arabidopsis thaliana*, called Patronus.<sup>111</sup> Although previous analyses did not reveal significant sequence similarity between human and yeast Securins,<sup>33</sup> the latest Pfam HMM profile (PF04856) encompasses both. To gather additional homologs, we searched for protein sequences containing the following motifs: a KEN box (lysine-glutamic acid-asparagine, which is involved in Anaphase-Promoting Complex/Cyclosome (APC/C)-mediated Securin proteolysis), a D-box (destruction box, which is also involved in APC/C-mediated Securin proteolysis), LPE (leucine-proline-glutamic acid, which blocks the interaction between Scc1 and Separase), SIS (separase-interacting segment, which inhibits Separase's protease activity by blocking substrate access to its active site).<sup>112-114</sup> Most candidate orthologs have an easily discernible KEN box and SIS motif. However, the D-box and LPE seem less conserved, and their positions within the protein seem to vary. Using iterative searches with jackhmmer,<sup>77</sup> we were able to connect animal and yeast Securin to plant Patronus proteins, thereby expanding the candidate ortholog set. Nonetheless, these searches also retrieved proteins like Cyclin(A/B) and Shugoshin due to their inclusion of KEN and D-box motifs. These proteins also have their own signature domains, which we searched for to exclude false Securin ortholog candidates.

Initially, we found Sororin to be metazoan-specific, but a recent paper presented compelling evidence on the homology of the C terminus of two candidate proteins (and others) in *A. thaliana* and *S. pombe* (Sor1),<sup>34</sup> specifically. This domain consists of two predicted alpha-helices in a region rich in both negatively charged amino acids (D/E) and bulky hydrophobic residues (FWL). To extend their observations we employed a strategy of profile sequence searches, whereby iterations of jackhmmer were used with different seed sequences. In case these jackhmmer iterations would either converge or share several hits along their trajectories, we concluded they would be homologous. During these iterations, we would also allow for the inclusion of so-called twilight zone hits. Such hits do not strictly fall within the category of statistically significant similarity, but would nonetheless be included because of their compelling similarities in protein length (Sororin is relatively short) and amino acid compositions. In addition, we generated various lineage-specific profile HMMs with which we searched for highly divergent sequences using hmmsearch -max (i.e., without any heuristic filter). We required a candidate ortholog to at least be able to reciprocate known orthologs (*A. thaliana*, fission yeast, human) upon several jackhmmer iterations or through a direct hmmsearch -max search. However, given the degenerated nature of the C-terminal domain/motif, it proved difficult to achieve such reciprocity and we were left with around 20 putative Sororin orthologs (e.g., in Discoba and Amoebozoa) that were not included in the final list.

For each protein, we provide the following data files in a GitHub repository (https://github.com/jolienvanhooff/SMCevolution, published release at Zenodo: https://doi.org/10.5281/zenodo.15373509).
(1) a text file containing the ortholog identifiers;

(2) a FASTA file containing unaligned orthologs;

(3) the profile HMM used to search for orthologs;

(4) the FASTA file containing the (curated) multiple sequence alignment for the profile HMM;

(5) the FASTA file containing the multiple sequence alignment used for phylogenetic inference;

(6) the logfile of the phylogenetic inference;

(7) the annotated gene phylogeny.

## Phylogenetic and network analysis in eukaryotes and prokaryotes

### Eukaryotic and prokaryotic dataset assembly

To study the pre-LECA origins of eukaryotic SMC complex subunits, we assembled two protein datasets: one for eukaryotes and one for prokaryotes. The eukaryotic dataset consists of a subset of the species we used to find orthologs in, which we subsampled to accelerate the analyses and increase the interpretability of the results, while still spanning eukaryotic diversity. The selected 49 species can be found in Table S1 (column 'PreLECASubset'). We annotated the orthologs of our proteins of interest, to facilitate the analyses and interpretation. The prokaryotic dataset was derived from GTDB (r207).<sup>81</sup> We used a subset of the assemblies in GTDB, ultimately including one assembly for each family-level clade. For each family, we selected assemblies that A), are the GTDB representative for a given species, B), have a CheckM<sup>82</sup> quality score (completeness score - 5"contamination score) above 50, and C), have the best CheckM quality score of their family. This resulted in a prokaryotic dataset containing 456 archaea and 3645 bacteria.

### Protein selection

We selected proteins that we deemed informative about the deep evolutionary origins of SMC complexes. We thereto applied the following criteria.

(1) The protein was present in LECA;

(2) The protein was present in an SMC complex in LECA as a permanent constituent, i.e., not as a regulatory or transient subunit;

(3) The protein belongs to a family of typical SMC complex subunits, and is not likely to be derived from other cellular machineries.

Based on these criteria we included the following proteins and their families: SMC1-6 (SMC protein family), CAPH, CAPH2, Scc1, Rec8 and Nse4 (kleisin family), CAPG, CAPD2, CAPG2, CAPD3, Scc3, PDS5 and NIPBL (hawk family), Nse1 and Nse3 (kite family), and Nse5 (family unknown) and Nse6 (family unknown). As part of our effort to find the evolutionary origins of the hawk family and Nse5.6, we searched for those of MAU2 and WAPL as well, in our HMM network analysis (see below).

### Phylogenetic analysis

We restricted the phylogenetic analysis to the SMC proteins, the kleisins and the kites, since we deemed the hawks, Nse5 and Nse6 too divergent for phylogenetic analysis. An overview of the applied phylogenetic methods for each family can be found in Table S2 (sheet 'Homologs'). In general, we used profile HMM of separate orthologous sets (SMC proteins) or, for less conserved proteins, a profile HMM based on their merged alignments (kleisin, kite) to first search across our eukaryotic dataset. With this first search, we confirmed that there are no obvious other eukaryotic homologs beyond the proteins making up the SMC complexes.<sup>10</sup> Then, we searched iteratively across our prokaryotic sequence dataset, and collected significant prokaryotic hits. We retrieved the eukaryotic protein sequences from the ortholog sets, i.e., those belonging to species of the subsampled dataset, and combined these with the prokaryotic hits. To allow for phylogenetic inference using a relatively complex substitution model (see below), which is computationally more intense, we further downsampled the SMC and kleisin sequences based on sequence identity with CD-HIT (v4.8.1, different identity cutoffs, Table S2, sheet 'Homologs').<sup>83</sup> For kite, such downsampling was not necessary, because for this family we limited the phylogenetic analyses to eukaryotic sequences and their readily discernible homologs in archaea, due to which the total number of sequences was lower. We chose to exclude bacterial kites, as well as archaeal kites that we were not able to recruit with HMM-based sequence searches, since these were likely to be too distinct from the eukaryotic sequences to meaningfully align them, or to model the evolution of in the phylogenetic inference.

The remaining protein sequences were aligned using MAFFT-L-INS-i or MAFFT-DASH<sup>72</sup> (v7.515), or using the PROMALS3D web server.<sup>85</sup> We selected the alignment approach yielding the most positions after trimming, as well as the best phylogeny (discussed below). For PROMALS3D, we obtained predicted protein structures from diverse sequences from the AlphaFold Protein Structure Database,<sup>79</sup> and included them as input. We trimmed the resulting multiple sequence alignments (MSAs) with trimAl in the 'gappyout' mode (v1.4.1)<sup>84</sup> and manually cut out regions not well-conserved. We cleaned the MSA from sequences that consisted of over >70% of gaps. Phylogenetic analysis was implemented through inference of a maximum likelihood (ML) phylogeny with IQ-TREE (v.2.0.3),<sup>36</sup> using the site-heterogeneous LG + C60 + G substitution model and assessing branch support using ultrafast bootstrap approximation (UFBOOT,<sup>37</sup> 1000 replicates). To test the topology of the resulting phylogenies, we inferred another phylogeny with the same model, constraining the eukaryotic sequences to be monophyletic. We compared the original tree (the ML tree or, if it had a higher likelihood, the consensus tree) to the constrained tree to assess if the constrained tree can be rejected using the approximately unbiased (AU) test provided by IQ-TREE.

For all families, we attempted to use PROMALS3D for generating an MSA, because it, in theory, provides a means to improve the alignment of divergent sequences, since the sequence alignment is guided by the alignment of (more conserved) structures. While
this worked well for the SMC proteins, it did not for the kleisin and kite families, as subsequent phylogenetic inference failed to recover the individual eukaryotic orthologous groups. Sequence members of these orthologous groups were not monophyletic, while they should have been. Moreover, ultrafast bootstrap support values were low for various key bipartitions. Possibly, these poor signals are resulting from a flawed structural guide alignment, although we did not further examine the potential underlying causes. For kites, we were able to use some structural information by employing MAFFT-DASH, which recruits resolved protein structures from the PDB, based on sequence similarity. Since it does not use predicted protein structures, the number of structures used as guides is low. For kleisins, generating a good MSA turned out particularly challenging. Therefore, we applied MAFFT-L-INS-i to the individual orthologous groups (except for Scc1 and Rec8, which were combined into a single alignment, because they were interspersed in previous phylogenies), and subsequently merged them using the 'merge' option, also adding the prokaryotic homologs.

PROMALS3D alignment-based phylogenies were pruned to remove leaves that corresponded to input structures. Furthermore, the SMC phylogeny, in the final visualization in Figure 4, was pruned from four leaves with very long branches (>5 average substitutions per site), and with unclear phylogenetic affiliations, since these might comprise spurious sequences. The clades were annotated according to A), their membership of a eukaryotic orthologous group (eukaryotic sequences) and B, their sisterhood relationship to the eukaryotic orthologous groups (prokaryotic sequences). If their relationship to eukaryotic sequences was not clear, we labeled them as 'unknown' (not necessarily monophyletic), or, in the case of the SpcB-like kites, as 'ScpB-like'. The distributions of each group across archaea are provided in Table S3. Note that, for SMC and kleisin proteins, these distributions form an underrepresentation due to the downsampling of the sequences for the phylogenetic analysis, as described above.

To specifically explore the relationships among the condensin/cohesin kleisins (CAPH, CAPH2, Scc1 and Rec8), we inferred an additional, more restricted phylogeny. We collected prokaryotic homologs based on a profile HMM constructed with a MAFFT-L-INS-i alignment of these four proteins. Then, we aligned the eukaryotic and prokaryotic sequences using MAFFT-L-INS-i and 'merge' as described above, trimmed the alignment with trimAl 'gappyout', removed sequences with >70% gaps, and applied IQ-TREE (substitution model: LG + C60 + G, 1000 UFBOOT replicates). Note that in the kleisin tree, four sequences with very long branches (>5 average substitutions per site) and unclear affiliations were removed.

For the phylogenies inferred for the SMC, kleisin and kite proteins, we shared on our GitHub repository (https://github.com/jolienvanhooff/SMCevolution, published release at https://doi.org/10.5281/zenodo.15373509)

(1) The unprocessed multiple sequence alignment.

(2) The multiple sequence alignment used as input for phylogenetic inference.

(3) The maximum likelihood and the consensus phylogenies.

(4) The logfile from the maximum likelihood phylogenetic inference.

(5) The maximum likelihood phylogeny inferred with constraint topology, i.e., with eukaryotic sequences forced to form a monophyly.

(6) The reporting file of the topology test.

In addition, we share the unfiltered, uncollapsed, annotated phylogenies corresponding to Figures 4A–4C on the iTOL web server.<sup>115</sup> Hyperlinks can be found in the legend of Figure 4.

## Constructing the profile-versus-profile HMM network

Reconstructing the evolutionary history of the hawk proteins, Nse5,6, WAPL, and MAU2 proved challenging using classical homology detection and phylogenetic inference methods due to their high sequence divergence, as noted before.<sup>5</sup> To address these difficulties, we employed the profile-versus-profile HMM-based network approach introduced by.<sup>5</sup> Briefly, we constructed a target profile database consisting of the MSAs generated here and combined this with the precomputed COG_KOG_v1.0 database (downloaded from http://ftp.tuebingen.mpg.de/pub/ebio/protevo/toolkit/databases/hhsuite_dbs/COG_KOG.tar.gz, last accessed Jan 17, 2023). We queried our manually-curated MSAs against this target database using the HHsearch algorithm from the HH-suite package<sup>86</sup> (v3.3.0) with default parameters as implemented in the MPI Bioinformatics Toolkit.<sup>78,86</sup> Following this initial search, all KOG/COG profiles that were hit by a query profile (E < 1000) in this first step were then used to complete reciprocal searches on the same database. The resultant graph (Table S5) was visualized using Cytoscape.<sup>88</sup> Here, we first imposed thresholds for the E-value (<1e-3) and alignment length (≥100), to keep only the edges that imply meaningful significant similarity between profiles. Then, Markov Chain Algorithm (MCL) clustering was performed with an inflation parameter *I* = 3 using 1/E-value as array source as implemented in the clusterMaker2 package.<sup>87</sup> Finally, we used Cytoscape's Prefuse Force Directed Layout with default settings, except for iterations = 1000, default node mass = 10, and we set the 'Force deterministic layouts' to 'on' and did not partition the graph before applying the layout. We also calculated the 'connectivity' between clusters. Connectivity is defined as the number of observed edges going from cluster 1 to cluster 2 (E-value=<1e-3 and alignment length>=100) over the total number of possible edges (Table S6).

## WAPL classification

Our profile-versus-profile HMM network revealed WAPL to be more closely related to ARM repeat proteins instead of HEAT repeat proteins (Figure 5A). ARM and HEAT repeats, while homologous and difficult to distinguish, represent distinct evolutionary families. A key structural distinction is that ARM repeat units typically consist of three helices, whereas HEAT repeat units typically contain two.<sup>116,117</sup> The published structures of WAPL orthologues from human (PDB: 4K6J)<sup>40</sup> and the fungus *Ashbya gossypii*
(PDB: 3ZIK)⁴¹ show three helices per unit. We used these structures to query the human AFDB-PROTEOME database with Fold-seek⁸⁰ and identified a strong enrichment of ARM-containing structures among the best hits (Table S4).

### Hawk structural alignment

Predicted 3D structures of hawks from both *A. thaliana* and *H. sapiens* were directly downloaded from the AlphaFold repository (alphafold.ebi.ac.uk/, last accessed 23 December 2023). Structures were manually checked in PyMOL⁹⁰ and N- or C-terminal disordered regions and/or other domains that do not pertain to the core HEAT-repeat region were removed (see Table S7 for coordinates and UniProt identifiers). The DALI webserver⁴² was used for structural comparisons among the hawks, using the all-against-all function. Z-scores were clustered with average linkage clustering using the Pearson coefficient as a distance measure (D = 1-r; Table S7). A PyMOL session is included in the GitHub repository (https://github.com/jolienvanhooff/SMCevolution, published release at https://doi.org/10.5281/zenodo.15373509).

### Nse5,6 alpha-solenoid structure prediction, alignment, and classification

To get a comprehensive view of Nse5 and Nse6 structures across eukaryotic diversity, we used the AlphaFold2 software to predict the structures of our obtained set of Nse5 and Nse6 orthologs.⁹⁶ Whereas for some orthologs experimentally determined structures are available, this is only the case for a fraction of the orthologs and species, while comparing their structures across eukaryotic diversity is needed to avoid lineage-specific patterns of these rapidly evolving sequences dominating the signal. Moreover, predicted structures, in contrast to many experimental structures, are complete along the protein's length. For these reasons, our structure comparisons (Figure S2) were based on predicted structures only. AlphaFold2 was run through the local ColabFold implementation.⁹⁷ We used alignments produced by MAFFT-L-INS-i of our curated sets of orthologs as input to ensure deep alignment information to produce high-quality structure predictions. Subsequently, the alpha-solenoids were isolated from the full-length structures using the pdb-tools package.⁹⁸ The start and end coordinates of the alpha-solenoids of each full-length structure were determined by aligning them to manually curated human Nse5 or Nse6 alpha-solenoids using Foldseek.⁸⁰ All isolated Nse5 and Nse6 alpha-solenoids were then comprehensively aligned in a pairwise fashion using TM-align,⁸⁹ where a TM-score of ≥0.5 was considered the threshold for significance.⁴⁷

To investigate to which group of alpha-solenoids Nse5 and Nse6 belong, their sequences and predicted structures were used. The N-terminal repeats of the Nse5 alpha-solenoid show homology to TPR/PPR, rather than HEAT. The remainder of the Nse5 and the complete Nse6 alpha-solenoids show twilight-zone (1000>E-value>0.001) hits for hawk proteins (Table S5). The results are, due to their tremendous divergence, inconclusive about the alpha-solenoid type of Nse5 and Nse6, and we leave open the possibility that they consist of mergers of different tandem repeat types.

### Visualization

The simplified species tree (Figures 2 and 6), the distributions of SMC complex subunits across eukaryotes (Figure 3) and the protein family phylogenies (Figure 4) were drawn using iTOL.¹¹⁵ Phylogenies were edited and iTOL input datasets were generated using ETE (v3)⁹³ and BioPython (v1.79).⁹⁴ Structures for Figure 5 were visualized and edited using PyMOL.⁹⁰ Structures for Figure S2C were visualized using UCSF ChimeraX.⁹² The heatmap in Figure 5B was visualized using R v4.2.3. All final figures were prepared using Adobe Illustrator.

## QUANTIFICATION AND STATISTICAL ANALYSIS

We visualized the distributions of the pairwise TM-scores of Nse5 and Nse6 orthologs as violin plots in Figure S2B, using R v4.2.3 and ggplot2. We included the number of alignments for each group (n) in the figure. No inferential statistics were applied to these distributions. We calculated their descriptive statistics using R v4.2.3, and the corresponding values are reported in the figure legend of Figure S2B.