bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1

2

3

4 Gradual evolution of cell cycle regulation by cyclin-

5 dependent kinases during the transition to animal

6 multicellularity

7

8

9 Alberto Perez-Posada1, Omaya Dudin1, Eduard Ocaña-Pallarès1, Iñaki Ruiz- 10 Trillo1,2,3*, Andrej Ondracka1*

11 1 Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra), Passeig 12 Marítim de la Barceloneta 37-49, 08003 Barcelona, Catalonia, Spain

13 2 Departament de Genètica, Microbiologia i Estadística, Universitat de 14 Barcelona, Av. Diagonal, 645, 08028 Barcelona, Catalonia, Spain

15 3 ICREA, Passeig Lluís Companys 23, 08010, Barcelona, Catalonia, Spain

16

17 *Corresponding authors

18 Email: [email protected]

19 Email: [email protected]

20

1 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

21 Abstract

22 Progression through the cell cycle in eukaryotes is regulated on multiple levels. 23 The main driver of the cell cycle progression is the periodic activity of cyclin- 24 dependent kinase (CDK) complexes. In parallel, transcription during the cell cycle 25 is regulated by a transcriptional program that ensures the just-in-time gene 26 expression. Many core cell cycle regulators are present in all eukaryotes, among 27 them cyclins and CDKs; however, periodic transcriptional programs are divergent 28 between distantly related species. In addition, many otherwise conserved cell 29 cycle regulators have been lost and independently evolved in yeast, a widely 30 used model organism for cell cycle research. To gain insight into the cell cycle 31 regulation in a more representative opisthokont, we investigated the cell cycle 32 regulation at the transcriptional level of Capsaspora owczarzaki, a species 33 closely related to animals. We developed a protocol for cell cycle synchronization 34 in Capsaspora cultures and assessed gene expression over time across the 35 entire cell cycle. We identified a set of 801 periodic genes that grouped into five 36 clusters of expression over time. Comparison with datasets from other 37 eukaryotes revealed that the periodic transcriptional program of Capsaspora is 38 most similar to that of animal cells. We found that orthologues of cyclin A, B and 39 E are expressed at the same cell cycle stages as in human cells and in the same 40 temporal order. However, in contrast to human cells where these cyclins interact 41 with multiple CDKs, Capsaspora cyclins likely interact with a single ancestral 42 CDK1-3. Thus, the Capsaspora cyclin-CDK system could represent an 43 intermediate state in the evolution of animal-like cyclin-CDK regulation. Overall, 44 our results demonstrate that Capsaspora could be a useful unicellular model 45 system for animal cell cycle regulation.

46 Keywords

47 cell cycle, evolution of cell cycle, periodic gene, transcriptional program, cell 48 division, synchronization of cell cultures, opisthokonta, holozoan

49

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50 Author’s summary

51 When cells reproduce, proper duplication and splitting of the genetic material is 52 ensured by cell cycle control systems. Many of the regulators in these systems 53 are present across all eukaryotes, such as cyclin and cyclin-dependent kinases 54 (CDK), or the E2F-Rb transcriptional network. Opisthokonts, the group 55 comprising animals, yeasts and their unicellular relatives, represent a puzzling 56 scenario: in contrast to animals, where the cell cycle core machinery seems to 57 be conserved, studies in yeasts have shown that some of these regulators have 58 been lost and independently evolved. For a better understanding of the evolution 59 of the cell cycle regulation in opisthokonts, and ultimately in the lineage leading 60 to animals, we have studied cell cycle regulation in Capsaspora owczarzaki, a 61 unicellular amoeba more closely related to animals than fungi that retains the 62 ancestral cell cycle toolkit. Our findings suggest that, in the ancestor of 63 Capsaspora and animals, cyclins oscillate in the same temporal order as in 64 animals, and that expansion of CDKs occurred later in the lineage that led to 65 animals.

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66 Introduction

67 The cell cycle is an essential and fundamental biological process that 68 underpins the cell division and proliferation of all cells. Progression through the 69 cell cycle involves multiple layers of regulation [1]. The main regulatory networks 70 that govern the transitions between cell cycle stages are broadly conserved in 71 eukaryotes, both on the level of individual regulators [2], as well as on the level 72 of network topology [3]. However, it is still not well understood how this conserved 73 regulatory network is deployed in cells with different cellular lifestyles and how it 74 changes across evolution.

75 Among the main regulators of the progression through the cell cycle are 76 cyclins and cyclin-dependent kinases (CDKs), two gene families broadly 77 conserved across eukaryotes [1,2]. Cyclins and CDKs have undergone 78 independent expansions and subfunctionalization in every major lineage of 79 eukaryotes, including opisthokonts [4–6]. In animals, there are multiple cyclins 80 and CDKs that form discrete complexes, activating specific downstream effectors 81 in different phases of the cell cycle [7,8]. Cyclin D-CDK4,6 complexes control 82 entry into the cell cycle in response to mitogenic factors [9–11]. The G1/S 83 transition is driven by the Cyclin E/CDK2 complex [12,13], and progression 84 through S phase is controlled by the Cyclin A/CDK2 complex [13]. Lastly, cyclin 85 B/CDK1 drive completion of mitosis [14,15]. In contrast, in the budding yeast 86 Saccharomyces cerevisiae one single CDK sequentially binds to nine cyclins in 87 three temporal waves [16,17]: Cln1-2 are expressed in G1 and mark the 88 commitment to a new cycle [18–21], Clb5,6 promote DNA replication at S phase 89 [22,23], and Clb1-4 drive progression through mitosis [24,25]. The fission yeast 90 Schizosaccharomyces pombe has a single CDK that also binds different cyclins 91 at G1,S, and M: Cig1,2 drive progression through G1 and S phase [26,27], and 92 Cdc13 drives progression through mitosis [28,29]. However, a single CDK-Cyclin 93 complex can drive progression through the entire cell cycle in this species [30].

94 In addition to the cyclin-CDK activity, the cell cycle is also regulated at the 95 transcriptional level by timing the expression of genes required in its different 96 phases. For instance, the E2F-Rb network of transcription factors controls 97 initiation of DNA replication in animals at the G1/S transition [31–33]. In yeasts,

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98 transcriptional regulation of the G1/S transition is driven by SBF and MBF, two 99 transcription factor complexes that bear no homology to E2F [34]. Recent findings 100 show that these transcription factors were acquired through lateral gene transfer 101 during fungal evolution [2]. In addition, other transcription factors are the mitotic 102 Fkh1 and Fkh2 [35,36] in S. cerevisiae or the Hcm1 transcription factor, 103 controlling progression through G2 and mitosis [37]. In human cells, a protein of 104 the same family, FoxM1, also regulates gene expression in mitosis [32,38]. 105 Although oscillatory transcriptional activity during the cell cycle is present in 106 numerous species and cell types [32,33,46–50,37,39–45], the genes affected by 107 cell cycle-regulated transcription are divergent between distantly related species 108 [51]. Likewise, even among different human cell types, periodic expression of 109 only a fraction of genes is common to all of them [32,52].

110 Yeasts have historically been a powerful model system to understand the 111 control of the cell cycle in animals. However, it has become clear that many 112 otherwise conserved cell cycle regulators have been lost and independently 113 evolved in the fungal lineage [2,3,53–55]. Thus, we sought to investigate the cell 114 cycle control in another organism within opisthokonts that has retained the 115 ancestral cell cycle regulation. We focused on Capsaspora owczarzaki (hereafter 116 Capsaspora), a species more closely related to animals than yeasts, easy to 117 culture, and for which good genomic resources are available [56–59]. This 118 amoeba has a life cycle that includes three distinct stages that differ both in their 119 morphology and transcriptional and proteomic profiles: amoebas with filopodia 120 that proliferate in adherent cultures, an aggregative multicellular stage in which 121 cells produce an extracellular matrix, and a cystic form that lacks filopodia [60– 122 62]. Moreover, Capsaspora has a compact, well-annotated genome, with many 123 homologs to animal genes [59]. With recent advances that allow transfection in 124 the laboratory [63], Capsaspora is becoming a tractable model organism.

125 In this work, we have established a protocol to synchronize cell cycle 126 progression in Capsaspora and have characterized its cell division and 127 transcriptional profile across the entire cell cycle. We found that globally, the 128 periodic transcriptional program of Capsaspora is enriched in genes that date 129 back to eukaryotic origin, and it resembles human cells more than the periodic 130 transcriptomes of yeasts. Out of four human cyclin types, Capsaspora contains

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131 homologs of cyclins A, B, and E. We found that these three cyclins are 132 transcriptionally regulated during the cell cycle and have a conserved temporal 133 order and cell cycle stage with human cells. In contrast, Capsaspora only 134 contains one ancestral copy of the CDK, which likely form complexes with all of 135 the Capsaspora cyclins. We also found that orthologs of many other cell cycle 136 regulatory genes have a conserved timing of expression compared with animal 137 cells. Our findings suggest that the cyclin-CDK system of animals evolved 138 gradually, through an intermediate stage where one single CDK was able to 139 interact with several cyclins at distinct stages of the cell cycle. Thus, while 140 expansion and subfunctionalization of animal cyclins occurred earlier, expansion 141 of CDKs occurred concomitantly with the emergence of animals.

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142 Results

143 Synchronization of cell cultures in Capsaspora

144 Synchronization of cell cultures is a basic experimental tool required to 145 study the cell cycle [64]. Cell synchronization methods include temperature- 146 sensitive strains, elutriation, cell sorting and commercially available inhibitors that 147 arrest the cell cycle. Arrest and release approaches have previously been used 148 to assess cell cycle progression in several organisms [39,42,65,66]. 149 Hydroxyurea, a widely used S-phase inhibitor in yeast cells [67], was already 150 shown to inhibit cell proliferation in Capsaspora cultures during the adherent cell 151 stage [62]. Therefore, to check if cell cycle inhibition occurred before entering S 152 phase and was reversible, we treated Capsaspora adherent cultures with 153 hydroxyurea and assessed DNA content by flow cytometry. Upon hydroxyurea 154 treatment, cells exhibited 1C DNA content, indicating arrest in G1 phase (Fig. 155 1A). Upon wash and release into fresh media, we observed synchronous 156 progression through the cell cycle as assessed by DNA content (Fig. 1C). This 157 indicates that hydroxyurea inhibits the cell cycle in S phase in Capsaspora and 158 that its effect is reversible.

159 To measure the timing of cell cycle stages in Capsaspora, we treated two 160 biological replicates of adherent proliferative Capsaspora cultures with 161 hydroxyurea, and we later released them into fresh medium. Samples were taken 162 at 45-minute intervals, starting from 2 hours after release, taking a total of 16 time 163 points that were analyzed using flow cytometry (Fig. 1B). Following release, cells 164 spent approximately 1.5 hours duplicating their DNA content, and after 8 hours 165 from release a G1 peak appeared again, indicating completion of the cell cycle 166 (Fig. 1C). These observations were reproduced in the two replicates. At later time 167 points, we noticed co-occurrence of 1C and 2C peaks. This may be due to some 168 cells progressing through the cell cycle more rapidly than others [68], causing 169 loss of synchrony, or due to an irreversible arrest in a fraction of the cells upon 170 HU treatment [69]. Nevertheless, our time course encompasses a complete 171 round of the cell cycle, as we observed an increase in 1C at later time points.

172

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173 Dynamics and morphology of cell division in Capsaspora

174 To characterize the dynamics of cell division in Capsaspora, we used time- 175 lapse microscopy in synchronized cells. In parallel, we analyzed synchronized 176 cells for DNA content by flow cytometry and characterized the cell morphology 177 during cell division using fluorescence microscopy. Out of a total of 100 mitotic 178 cells observed by live imaging (Fig. 2A), the highest fraction underwent 179 cytokinesis at approximately 10 hours (Fig. 2C). Cells seemed to round up and 180 slightly detach from the plate surface while retaining their filopodia (Fig. 2A, Video 181 1). This phenomenon has been previously characterized in other eukaryotic cells 182 lacking a rigid cell wall, such as animal cells or Dictyostelium amoeboid cells [70– 183 77]. Cells took an average of 3 minutes to completely undergo cytokinesis (Fig. 184 2A, Video 1), measured as the time from rounding up to the splitting of two 185 daughter cells. This value is at least twice as fast as in animal cell types, where 186 the average time for cytokinesis is 6 minutes [78–80], and it is also considerably 187 faster than in yeasts [81–84] when considered in relation to the total generation 188 time of Capsaspora (estimated to be around 12 hours in our culture conditions). 189 As shown in Fig. 2D, the measured area of daughter cells is roughly the same, 190 suggesting that cell division is symmetric and yields two equally sized cells.

191 To investigate the morphology of mitotic cells, we stained tubulin and DNA. 192 On each cell, we observed one dot of dense concentrations of tubulin, from which 193 microtubules emanate (Fig. 2B, white arrows). These dots duplicated and 194 remained close, first associated with the nucleus and then surrounding densely 195 packed DNA. A central, thicker spindle emerged and grew as DNA separated and 196 moved to opposite poles of the dividing cell. A similar phenomenon has been 197 described for Dictyostelium [85]. Previous studies have reported the absence of 198 proteins from the gamma-tubulin ring complex and the yeast spindle pole body in 199 Capsaspora [86], which together with our observations suggests that Capsaspora 200 mitotic spindle is organized without a microtubule organizing center (MTOC), or 201 that it possesses an independently evolved MTOC, such as in yeast [87].

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202 Detection of periodically expressed genes during the Capsaspora cell 203 cycle

204 In many cell types, cell division cycles are accompanied by a transcriptional 205 program of periodic gene expression over time. To understand the transcriptome 206 dynamics during the cell cycle of Capsaspora, we performed time-series RNA- 207 seq experiments. We used RNA extracts from the same two biological replicates 208 as in the flow cytometry assay and sequenced them using Illumina HiSeq v4. We 209 processed the sequencing reads using Kallisto [88] (Supplementary File 1). 210 Spearman correlations by gene expression profiles showed that time points are 211 grouped according to the temporal order of sampling (Supplementary Fig. S2). 212 This indicates that gene expression is not shifted over time and was reproducible 213 between the two replicates. To detect periodicity patterns in gene expression, we 214 applied two algorithms, JTK_CYCLE [89] and RAIN [90], on an average dataset 215 of the two replicates where non-expressed genes were filtered out 216 (Supplementary Fig. S1A). We assigned two ranks to every gene according to 217 the p-values calculated by JTK_CYCLE and RAIN (Fig. 3B), and assigned the 218 final periodicity rank as the sum of JTK and RAIN ranks. We applied a 219 conservative cutoff to identify genes that are undoubtedly periodically transcribed 220 by taking the top 800 genes ranked within the top 2000 ranking for each 221 independent dataset (Fig. 3B) (Supplementary Fig. S1B). This cutoff corresponds 222 to 10% of the total number of genes in Capsaspora, a fraction similar to those 223 observed in other species [39,42,50,91]. Although false negatives with higher 224 ranks might have been discarded due to our conservative approach, we 225 confirmed that top-ranked genes showed oscillatory behavior. We manually 226 included Capsaspora Cyclin A (CAOG_04719T0) [4,59] (see below) to the list of 227 periodic genes despite ranking in position 1916, as it has a periodic expression 228 profile (Fig. 5A).

229 For a gene expression dataset containing only genes identified as periodic, 230 we observed stronger correlations by time points (Fig. 3C) or pairwise genes than 231 for the entire dataset (Supplementary Fig. S3A). We also observed a strong 232 correlation between initial and late time points (Fig. 3C), suggesting that the 233 cultures indeed completed the entire cell cycle despite the loss of synchrony. A 234 principal coordinate analysis using data for the top-ranked periodic genes

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235 retrieved a grouping of time points that resembles a circle with two components 236 explaining around 70% of the total distance (Fig. 3D, Supplementary Fig. S3B), 237 and a similar layout is obtained on a t-SNE plot of periodically expressed genes 238 (Supplementary Fig. S4). The results from these analyses indicate that there is a 239 set of periodically transcribed genes during the cell cycle of synchronous 240 Capsaspora cells. This set of genes represents the periodic transcriptional 241 program of Capsaspora (Supplementary File 2).

242 Gene expression is clustered in periodic waves during the cell cycle

243 The cell-cycle-associated transcriptional program responds to the 244 requirements of the cell at a given moment. For example, in many cell types, 245 genes necessary for DNA replication or mitosis are transcribed only at the time 246 of their biochemical activity. The detection of periodic genes in Capsaspora 247 prompted us to classify them into temporal clusters, by centering their expression 248 profiles to the mean and grouping them using hierarchical clustering based on a 249 dissimilarity matrix by Euclidean distance (Fig. 4A). Five clusters were detected 250 according to the similarity in expression profiles over time (Fig. 4A, 251 Supplementary Fig. S4A, Supplementary Fig. S4B), and we obtained very similar 252 results by using k-means clustering (Supplementary Fig. S5). We then associated 253 these gene clusters to the cell cycle stage during the peak of expression: the 254 G1/S cluster, S cluster, G2/M cluster, M cluster, and G1 cluster. Next, we 255 calculated gene ontology (GO) enrichment for every cluster against the whole 256 periodic transcriptional program using Ontologizer [92] Parental-Child-Union 257 calculation (Fig. 4B, Supplementary File 3).

258 The G1/S cluster contains 194 genes peaking in the initial time points, from 259 2 to 3.5 hours after release. Genes found here exhibit the largest differences in 260 gene expression between time points and are enriched in GO terms related to 261 DNA replication, deoxyribonucleotide biosynthesis, and chromosome 262 organization. There is also enrichment in the GO term “response to stress”, 263 suggesting an effect of the treatment with hydroxyurea [68,69]. The small cluster 264 assigned to S/early G2 contains 36 genes peaking between 3.5 to 5.75 hours 265 enriched in the GO term “nucleosome binding” and includes several histone 266 genes.

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267 In the G2/M cluster, we found 176 genes with the peak of expression at 6.5- 268 7.25 hours post-release. This cluster is enriched in the GO term “non-coding RNA 269 metabolic process”, and contains genes related to tRNA maturation such as 270 RTCB, DDX1 [93], and tRNA ligases. It has been previously reported that tRNA 271 synthesis can increase during the cell cycle in several systems [94–96]. To our 272 knowledge, however, there is still no link reported between tRNA modification 273 and progression through the cell cycle.

274 The M cluster is the largest one in the dataset, with 241 genes peaking 275 between 9.5 and 11 hours. Genes reach the highest expression during time 276 points when the cells enter mitosis, reaching a plateau which reflects the partial 277 asynchrony of the cells at the time. This cluster is enriched for genes annotated 278 with chromosome segregation, organelle fission, and diverse cytoskeletal 279 components like spindle proteins and myosins. All these GO terms can be linked 280 to mitotic cell division.

281 The G1 cluster has 157 genes. Genes in this cluster show higher expression 282 levels in both the late and initial time points of the experiment. Many GO terms 283 enriched in this cluster are related to the mitochondrion and diverse metabolic 284 processes that indicate an increase in cell metabolism as the cell progresses 285 through the cell cycle [97].

286 Taken together, the GO enrichment analyses show that gene expression 287 clusters contain conserved genes involved in the cell cycle in Capsaspora.

288 Conserved temporal order of cyclin and CDK expression in 289 Capsaspora

290 In eukaryotes, the cell cycle events are regulated by cyclins in complex with 291 CDKs. While the cyclin and CDK gene families are broadly conserved across 292 eukaryotes [2,3], some of the subfamilies are lineage specific and have radiated 293 differently. In budding yeast, two types of cell cycle cyclins can be found: cyclin 294 B and Cln-type cyclins. Both types bind to one single CDK, Cdk1 [16]. In animals, 295 this ancestral CDK expanded and specialized [4] resulting in multiple cyclin-CDK 296 partners involved in different phases of the cell cycle: CDK2 binds to cyclins E 297 and A at the onset and later stages of S phase [13], cyclin B binds to Cdc2 in

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298 mitosis [14], and CDK4 and CDK6 bind to cyclin D types during G1 [98]. To our 299 knowledge, how cyclin-CDK binding partnership grew in complexity remains 300 unclear.

301 To provide some insights into the evolution of cyclin-CDK binding 302 partnership, we used our temporal gene expression dataset to infer the timing of 303 cyclin-CDK activity during the cell cycle in Capsaspora. Due to inconsistencies in 304 published work [4,59], we first revised the classification of cyclin and CDK genes 305 found in Capsaspora by phylogenetic profiling using a complete taxon sampling 306 for Holozoa (the clade comprising animals and their closest unicellular relatives) 307 and validated the gene previously reported as Capsaspora CDK1 [59] by Sanger 308 sequencing and transcriptomics (see Methods) (Supplementary File 4, 309 Supplementary File 5, Supplementary Figs 6-8). Despite the limited phylogenetic 310 resolution, Capsaspora and other unicellular holozoan sequences appeared in 311 earlier branching positions to the metazoan Cyclin A, B and E clades, this being 312 compatible with Capsaspora having orthologs of these cyclins (Supplementary 313 Fig. S6). The phylogeny does not suggest the presence of a Cyclin D ortholog in 314 Capsaspora. In the CDK phylogeny (Fig. 5-Fig. supplement 2), sequences from 315 Capsaspora and other filastereans branch as a sister-group to the metazoan 316 CDK1 clade (100% of UFBoot), whereas the branching of sequences from other 317 unicellular holozoans is more uncertain concerning the CDK1 and CDK2-3 318 metazoan clades. From this, we envision two possible evolutionary scenarios. In 319 a first scenario, an ancestral duplication of CDK1-3 into CDK1 and CDK2-3 320 occurred in a common ancestor of Holozoa. As most unicellular holozoans have 321 only one sequence within the CDK1-3 clade, this would imply differential losses 322 of either CDK1 or CDK2-3 in Ichthyosporea, Filasterea and Choanoflagellatea, 323 and Metazoa conserving both paralogs. Despite Salpingoeca rosetta has two 324 sequences within the CDK1-3 clade, both paralogs are likely to descend from a 325 duplication event occurred in the Choanoflagellatea lineage, with Monosiga 326 brevicollis losing one of the two copies. In a second scenario, which we find more 327 parsimonious, the duplication would have occurred in the lineage leading to 328 Metazoa, but the limiting phylogenetic signal would not have allowed 329 reconstruction of the real phylogenetic pattern of the CDK1-3 clade. Thus, we 330 propose that the subfunctionalization of CDK1-3 is a specific feature of Metazoa,

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331 with Capsaspora retaining the ancestral CDK1-3 gene instead of having a CDK1 332 ortholog as previously reported [59].

333 We report a clear temporal ordering of expression of the putative 334 Capsaspora Cyclins A, B, and E (Fig. 5A). Cyclin E belongs to the G1/S cluster, 335 cyclin A clusters together with S-phase genes, and cyclin B is in the M cluster, 336 peaking at mitosis (Fig.4) together with Capsaspora CDK1-3 (Fig. 5B), although 337 we found the CDK1/2/3 transcript expressed at high levels also during the rest of 338 the cell cycle. In conclusion, our results suggest that cyclins A, B and E follow the 339 same temporal order and cell cycle phases as cyclins in human cells.

340

341 The Capsaspora periodic transcriptional program includes ancient 342 eukaryotic genes and is similar to that of animal cells

343 Besides the cyclin-CDK system, other regulators are periodically expressed 344 during the cell cycle [32,39,40]. To characterize the periodic expression program 345 in Capsaspora in comparison to other species, we identified orthologs of cell cycle 346 regulators from other species in Capsaspora using OrthoFinder [99] 347 (Supplementary File 5) and using a list of one-to-one Capsaspora-human 348 orthologs from a set of phylogenies of Capsaspora [61]. From these sources, we 349 identified which periodic human genes with known functions in the cell cycle (as 350 described in [32,39,40,100]) have also a periodic ortholog in Capsaspora. We 351 found that numerous DNA replication genes are upregulated in the G1/S cluster 352 in Capsaspora, including DNA polymerase subunits, replication factors, and 353 proteins CDC45 and PCNA (Fig. 5C). Among human genes that peak in mitosis, 354 we found Capsaspora orthologs of Aurora Kinase A (AURKA), protein regulator 355 of cytokinesis 1 (PRC1) and the anaphase-promoting complex (APC) subunit 356 UBE2S expressed in the G2/M cluster (Fig. 5D). In human cells, AURKA 357 regulates the assembly of the centrosome and the mitotic spindle [101], mitotic 358 cyclins and cohesins are degraded by the APC [1], and PRC1 regulates 359 cytokinesis by cross-linking spindle midzone microtubules [102]. Although we did 360 not find regulatory APC subunits CDC20 and CDH1 [103,104] among periodically 361 expressed genes in Capsaspora, the UBE2S peak in mitosis suggests that APC 362 activity might also be transcriptionally regulated during the cell cycle in

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363 Capsaspora. During M phase, we also observed upregulation of different 364 kinesins, microtubule motors with conserved function in the mitotic spindle, and 365 centromere proteins; a more detailed overview is provided in Supplementary File 366 7. We also identified Capsaspora periodic genes belonging to the same 367 orthogroups as other cell cycle regulators in human cells, but without a one-to- 368 one ortholog relationship (Supplementary File 8).

369 In addition to the examples above, we were interested in the similarities of 370 the periodic transcriptional program of Capsaspora with those of other eukaryote 371 species. First, to understand the evolutionary origin of the periodic genes found 372 in Capsaspora, we calculated gene age enrichment for every cell cycle cluster. 373 We assigned a gene age to the orthogroups by Dollo parsimony [105] and 374 compared the enrichment ratios for non-periodic genes and the five clusters 375 separately with the gene age of the whole transcriptome of Capsaspora. Three 376 of the clusters of periodic genes presented significant enrichment in pan- 377 eukaryotic genes (Fig. 6A, Fig. 6—source data 1). Our data thus shows that a 378 large fraction of genes in the periodic transcriptional program of Capsaspora 379 belong to gene families originating early in eukaryotic evolution.

380 Next, we compared our dataset of Capsaspora periodic genes with datasets 381 of cell cycle synchronized cells of different organisms, namely three different cell 382 types of Homo sapiens (Hela Cells, U2OS cells, and foreskin primary fibroblasts) 383 [32,41,50], Saccharomyces cerevisiae [43], Schizosaccharomyces pombe [91], 384 and Arabidopsis thaliana [44]. For each dataset, we took the published lists of 385 periodic genes and corrected for the number of genes in each species (Fig. 6— 386 source data 2). We set a threshold of less than 10% of genes to be periodic for 387 human cells and the yeasts, and less than 5% for A. thaliana. Thus 1790 periodic 388 genes were identified in HeLa cells, 1245 in U2OS cells, 461 in fibroblasts, 592 389 in S. cerevisiae, 499 in S. pombe, and 1060 in A. thaliana datasets. We found 390 1925 orthogroups that contained at least one periodic gene from either of the 391 datasets (Fig. 6B), and named these “periodic orthogroups”. Of these, one third 392 had orthologs in all five species.

393 We first computed the number of periodic genes shared between pairs of 394 species (defined by their presence in the same orthogroup). Overall, all species

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395 share a small number of periodic genes (Fig. 6—source data 3, Fig. 6—source 396 data 4), with the number of genes being highest for Capsaspora with H. sapiens 397 cell lines (167, 81 and 99 periodic orthogroups with HeLa cells, U2OS cells, and 398 fibroblasts, respectively) rather than yeasts or A. thaliana (Fig. 6D, 399 Supplementary File 11, Supplementary File 12). Still, the periodic expression of 400 the majority of the genes is not shared between species, consistent with former 401 findings of 2% to 5% of periodic genes shared between different organisms [51]. 402 This indicates that the periodic transcriptional program is divergent both between 403 species and even between different cell types within an organism.

404 Despite the low numbers of periodically expressed genes in common, we 405 wondered whether the periodic transcriptional program in each species indeed 406 evolved independently, meaning that the pairs of genes that share the periodic 407 expression between species are observed only by chance. To that end, we 408 calculated the expected number of shared periodic orthogroups by chance as the 409 product of ratios of periodic orthogroups from each species within their 410 orthogroups in common (Fig. 6C). We detected that, for most species, the number 411 of shared periodic genes is higher than by chance, especially for Capsaspora and 412 the core cell cycle gene set of H. sapiens (defined in [32] ) (Fig. 6D, 413 Supplementary File 11, Supplementary File 12). We found the same when 414 comparing periodic one-to-one orthologs [61] (Fig. 6D) or when defining periodic 415 genes on every species using the same method that we applied to Capsaspora 416 (Supplementary Fig. S9, Supplementary Files 10-12). Therefore, these findings 417 are robust with respect to the methods used to identify periodically expressed 418 genes and to assign orthology relationships. Thus, although the cell cycle 419 periodic expression program largely evolves fast and independently, our data 420 suggest there is a core set of genes of conserved oscillatory expression during 421 the cell cycle (Fig. 6B).

422 Overall, our cross-species comparison of the periodic gene expression 423 programs revealed that the Capsaspora periodic gene expression program is 424 more similar to human cells that to current unicellular model systems for the cell 425 cycle. Furthermore, including a new species in the global analysis, we discovered 426 a previously unappreciated core set of genes for which periodic expression is 427 deeply conserved.

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428 Discussion

429 In this study, we have used synchronized Capsaspora adherent cells to gain 430 insight into key aspects of the cell cycle, such as cell division and periodic gene 431 expression, in a unicellular relative of animals. Previously, the cell cycle has been 432 studied in only a handful of species due to the inability to obtain synchronous cell 433 cultures. With this synchronization protocol, the cell cycle of a closer relative of 434 animals can be studied in cultures that can be synchronized from DNA replication 435 to cell division.

436 Our experimental setup made possible to characterize mitotic cell division 437 in Capsaspora, which we found relies on microtubule-based structures, as 438 previously described in other eukaryote species (Forth and Kapoor, 2017). Our 439 observations suggest the presence of a putative non-centrosomal microtubule 440 organizing center (MTOC) in Capsaspora, which raises new questions about the 441 mechanisms of cell division in this species. As non-centrosomal MTOCs have 442 independently evolved in many different species [107], it may well be that 443 Capsaspora has a non-centrosomal, independently evolved MTOC, or that their 444 microtubules are able to self-arrange, as previously shown in other systems 445 [108].

446 Synchronization of Capsaspora cell cultures allowed us to study 447 transcription during the cell cycle using RNA sequencing. We identified five 448 waves of gene expression across time, with most genes being expressed in the 449 G1/S transition and in mitosis. As in previously studied organisms, these waves 450 of transcription can be grouped in clusters containing genes related to the main 451 events of the phases of the cell cycle. The periodic transcriptional program of 452 Capsaspora is enriched in genes that emerged at the onset of eukaryotes, 453 showing that the cell cycle relies in numerous genes, such as DNA replication 454 proteins and cytoskeleton components, which are common to all eukaryotes due 455 to their roles in fundamental cellular processes. Although transcriptional activity 456 during the cell cycle is present in numerous species and cell types [51], the genes 457 affected by cell-cycle-regulated transcription are divergent between distantly 458 related species, likely due to the fact that transcriptional regulation adapts to the 459 environment and lifestyle of each particular cell type. Our observations suggest

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460 that this occurs largely by old genes gaining and losing periodic regulation, rather 461 than new species-specific genes evolving to be regulated during the cell cycle. 462 This is consistent with Jensen et al.’s observations that periodicity in complex 463 activity can evolve rapidly in different lineages by recruiting different partners of 464 the same complex, which preserves the periodic regulation of the entire complex 465 [51]. Still, in contrast to previous analysis with a more limited set of species, our 466 analysis clearly revealed a core set of genes of which the periodic regulation is 467 deeply conserved among eukaryotes.

468 From the temporal order of gene expression of Capsaspora cyclins, we 469 conclude that cyclins A, E, and B follow the same order and are associated with 470 the same cell cycle stage as in H. sapiens cells. In contrast to human cells, where 471 these cyclins bind their respective partner CDKs, Capsaspora only possesses 472 one ancestral CDK1-3, which, although periodically expressed with a peak in M- 473 phase, exhibits high transcript levels throughout the cell cycle. Due to this, we 474 propose that CDK1-3 might be the binding partner of cyclin A, B, and E in 475 Capsaspora and that it might be involved in all phases of the cell cycle (Fig. 7). 476 Nevertheless, in the absence of biochemical data, we cannot exclude the 477 possibility that Capsaspora cyclins A and E bind other non-canonical CDKs. 478 Interestingly, in knock-out mice where all CDKs except for CDK1 were deleted, 479 all cyclins were also found to bind CDK1[109], which suggests that animal cyclins 480 A, B and E are also able to bind CDK1. Given these data, we reconstruct a likely 481 evolutionary scenario of the evolution of the cyclin-CDK system in opisthokonts. 482 The ancestral opisthokont likely possessed a single CDK, with a role in multiple 483 cell cycle phases, and B-type cyclins. Cyclins underwent duplication and 484 subfunctionalization first, acquiring roles in regulating distinct phases of the cell 485 cycle while binding to the single CDK. This evolutionary intermediate state is 486 present in Capsaspora. During the emergence of animals, CDKs also underwent 487 expansion and subfunctionalized to bind specific cyclins, forming discrete 488 complexes active in a particular stage. This suggests that there was a gradual 489 evolution of the cyclin-CDK control of the cell cycle during the emergence of 490 animals.

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491 Author contributions

492 A.P., O.D. and A.O. set up the methodology, and performed the time-series 493 experiment. A.P. performed the flow cytometry, analyzed the gene expression 494 data, sequenced the Capsaspora CDK1 gene, performed the comparative 495 analysis, and wrote the original draft. O.D. performed the microscopy. E.O.P. 496 performed the phylogenetic analysis. I.R.T. provided supervision. A.O. conceived 497 the project, performed the Capsaspora RNA extraction, and provided 498 supervision. All authors reviewed and edited the manuscript.

499 Acknowledgements

500 We thank all the members of Iñaki Ruiz-Trillo’s lab for discussion and 501 comments on the manuscript, Daniel Richter for discussion on the statistical 502 tests, Xavier Grau-Bové for helpful discussions on the data analysis, Arnau Sebé- 503 Pedrós for providing resources of the Capsaspora phylome, and Meritxell Antó- 504 Subirats for technical support. We also acknowledge the UPF Flow Cytometry 505 Core Facility for assistance with flow cytometry, and the CRG Genomics Unit for 506 mRNA library preparation and Illumina sequencing.

507 This work was funded by a European Research Council Consolidator Grant 508 (ERC-2012-Co-616960) to I.R.T; and a grant from the Spanish Ministry for 509 Economy and Competitiveness (MINECO; BFU2014-57779-P, with European 510 Regional Development Fund support) to I.R.T. A.P. was supported by a “la 511 Caixa” Foundation (ID 100010434) fellowship, whose code is 512 LCF/BQ/ES16/11570008. O.D. was supported by a Swiss National Science 513 Foundation Early PostDoc Mobility fellowship (P2LAP3_171815) and a Marie 514 Sklodowska-Curie individual fellowship (MSCA-IF 746044). E.O.P. was 515 supported by a pre-doctoral FPI grant from MINECO. A.O. was supported by a 516 Marie Sklodowska-Curie individual fellowship (MSCA-IF 747086). The funders 517 had no role in study design, data collection and analysis, decision to publish, or 518 preparation of the manuscript.

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519 Materials and methods

520 Cell cultures and culture synchronization

521 Capsaspora cells were incubated at 23ºC in ATCC medium 1034 (modified 522 PYNFH medium). Two independent cultures of Capsaspora at 30-50% 523 confluency were treated using 10mM Hydroxyurea (Sigma Aldrich, Saint Louis, 524 MO, USA, #H8627) in culture medium, and left incubating for approximately 14 525 hours. 526 527 Cells were released from Hydroxyurea by washing prior to elution in fresh 528 medium; samples were collected by scraping and washing two hours after 529 release, and from there on every 45 minutes until thirteen hours. A total of sixteen 530 time points were taken, constituting a time window comprising one event of 531 genome duplication and one mitotic division. 532 533 We also tested different concentrations and incubation times of 534 hydroxyurea, nocodazole (Sigma-Aldrich, #M1404), and aphidicolin (Sigma- 535 Aldrich, #A0781). Only 10mM hydroxyurea for longer than thirteen hours showed 536 arrest of the cell cycle, while Nocodazole had no observable effect by DNA 537 content measurement, and the rest ruined the samples due to insolubility of the 538 compound.

539 DNA content measurement

540 Cells were washed in PBS and fixed in 70% ethanol in PBS, then incubated 541 in RNAse A (Sigma-Aldrich, #R6148) (one volume in three volumes of 1xPBS) 542 for 24 hours at 37 ºC. Cells were then incubated in a final concentration of 543 20µg/ml propidium iodide (Sigma-Aldrich, #P4170-25MG) for 72 hours at 4 ºC. 544 545 Samples were analyzed by flow cytometry using a BD LSR Fortessa 546 analyser (Becton Dickinson, Franklin Lakes, NJ, USA). SSC-A and FSC-A were 547 used to detect populations of stained cells. Single cells were gated by FSC-H and 548 FSC-A. An average of 10,000 events per sample were recorded. PI-positive cells 549 were detected using a 561 nm laser with a 610/20 band pass filter (red

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550 fluorescence). To estimate the cell count, Texas Red-A was plotted as 551 histograms using FlowJo 9.9.3 (FlowJo LLC, Ashland, OR, USA).

552 Cell microscopy and image analysis

553 Microscopy pictures were taken using a Zeiss Axio Observer Z.1 554 Epifluorescence inverted microscope equipped with Colibri LED illumination 555 system and Axiocam 503 mono camera (Carl Zeiss microscopy, Oberkochen, 556 Germany). A Plan-Apochromat 100X/1.4 oil objective (Nikon Corporation, Tokyo, 557 Japan) was used for imaging fixed cells. For the live imaging, we used an EC 558 Plan-Neofluar 40x/0.75 air objective (Carl Zeiss microscopy). 559 560 Image analysis was done using ImageJ software [110]. For fixed cells, we 561 used the oval selection tool to draw the contour of each cell and measured cell 562 perimeter. As cells are spherical, we computed cell area using ImageJ. We 563 estimated the relative cell area of every pair of daughter cells by dividing each 564 measurement by the sum of the two daughter cells areas. All the calculation and 565 data plotting was done in R Software ver. 3.4.4[111].

566 RNA isolation and sequencing

567 Time point samples were washed in 1xPBS, poured in Trizol, and frozen at 568 -80ºC. Total RNA was purified using Zymo RNA miniprep kit (Zymo Research, 569 Irvine, CA, USA, #R2050). mRNA libraries were prepared using the TruSeq 570 Stranded mRNA Sample Prep kit (Illumina, San Diego, CA, USA, Cat. No. RS- 571 122-2101). Paired-end 50bp read length sequencing was carried out at the CRG 572 genomics core unit on an Illumina HiSeq v4 sequencer, with all samples from the 573 same replicate being pooled in the same lane. 574 575 Capsaspora adherent cultures cDNA was obtained by RT-PCR using 576 SuperScript® III Reverse Transcriptase (Invitrogen, Carlsbad, CA, USA, 577 #18080044) following the manufacturer’s instructions. PCR was performed using 578 Phusion® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA, 579 USA, #M0530L) following the manufacturer’s instructions.

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580 Transcriptomic analysis

581 RNA reads were mapped using Kallisto v0.43.1[88] using default 582 parameters onto a set of the largest isoforms of the Capsaspora 583 transcriptome[62]. The resulting time-series transcriptome in transcript-per- 584 million (tpm) units is available in Supplementary File 1. We retrieved only the 585 transcripts whose average expression level is above 1 tpm in the whole time 586 series. 587 588 Gene expression level was normalized by subtracting the mean over time 589 and dividing by the standard deviation. Normalized datasets were clustered 590 according to their Spearman correlation values using hierarchical clustering (R 591 gplots library[111,112]).

592 Identification of periodically expressed genes

593 Periodic genes were detected in Capsaspora by ranking using JTK_CYCLE 594 [89] and RAIN [90] on the time-series transcriptomes and an average of the two 595 replicates. We set JTK_CYCLE parameters to periods=14:16 and sampling 596 interval=0.75, and ranked every gene by their BH.Q value. We set RAIN 597 parameters to period=16 and delta=0.75, and ranked every gene by their 598 Benjamini-Hochberg corrected p-value. We set a cutoff of the genes ranked 599 below 2000 on each separate replicate and simultaneously ranked below 800 in 600 the average dataset (see Supplementary Fig S1, Supplementary File 2).

601 Clustering analysis

602 Periodic genes were hierarchically clustered according to similarity of gene 603 expression over time (averaged between two replicates), and clustered by k- 604 means clustering[111] using standard parameters and k=5. Agreement between 605 clustering methods was calculated as the number of genes belonging to the same 606 pair of clusters divided by the size of the smallest cluster in the pair.

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607 Calculation of Gene ontology enrichment

608 Gene ontology enrichment was calculated using Ontologizer[92] using the 609 –c “Parent-Child-Union” –m “Bonferroni” options. Bonferroni-corrected p-values 610 were taken as significant when below 0.05.

611 Phylogenetic classification of CDKs and Cyclins from early-holozoa 612 taxa

613 Annotated sequences of cyclins and CDKs from H. sapiens and S. 614 cerevisiae were retrieved from Cao et al.[4] and Swissprot[113], and A. thaliana 615 sequences were taken from[113–115]. These sequences were used as queries 616 to detect potential CDKs and cyclins orthologues in our dataset (Supplementary 617 File 4) using BLAST+ v2.3.0[116,117]. Those sequences that aligned were 618 BLASTed against a database including all proteins from H. sapiens, S. cerevisiae 619 and A. thaliana. We only included in our phylogeny those sequences whose best 620 hit against this database matched the original sequences used in the detection 621 step. Proteins were aligned with MAFFT v7.123b.[118], using the -einsi option, 622 and alignments were trimmed using trimAl v1.4.rev15[119] with the -gappyout 623 option. Trimmed alignments were manually inspected and cleaned of poorly 624 informative sequences except if that sequences corresponded to early-branching 625 holozoa (ebH) taxa. Cleaned alignment were used as inputs for phylogenetic 626 inference with IQ-TREE v1.6.7[120], using the -bb 1000 and -mset LG options. 627 For CDKs, since the tree topology did not show any ebH sequences belonging to 628 CDK families without orthologues in Metazoa, we performed a second 629 phylogenetic inference using only the ebH and metazoan proteins to reduce the 630 potential phylogenetic noise that may be introduced with the addition of non- 631 informative divergence. 632 633 Sequences were classified into CDK/Cyclins families taking into account the 634 tree topology and the UFBoot nodal support values[121]. Families were named 635 according to the orthology relationship between the ebH protein and the H. 636 sapiens sequence. For example, Sarc_g11690T was classified as CDK10 637 because its position in the tree suggests an orthology relationship to this H. 638 sapiens protein (Supplementary Fig. S7), whereas Cowc_CAOG_08444T0 was 639 classified as CDK11-CDK11B because it is orthologue to both H. sapiens

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640 proteins (Supplementary Fig. S7). We found three ebH cyclin subfamilies without 641 orthologues in Metazoa, which were named accordingly to the corresponding 642 orthologue in Saccharomyces cerevisiae (PCL1, PCL2, PCL9; PCL5, CLG1; and 643 PCL6-PCL7). Those ebH sequences showing ambiguous or poorly supported 644 branching patterns were classified as uncertain. 645 646 We found that the current Capsaspora sequence reported by[59] as a CDK1 647 orthologue (CAOG_07905) is considerably shorter in length than other CDK 648 orthologues. This could explain why it was not detected in the work by Cao et 649 al.[4], if an e-value constraint was taken into consideration. By inspection of the 650 genomic sequence from[122], we detected an assembly gap of 1kb neighboring 651 the 3’ end of the predicted annotation of CAOG_07905 (Supplementary Fig. 652 S8A). In silico translation of the surroundings of this gap revealed protein 653 domains conserved in H. sapiens and S. cerevisiae CDK1 proteins 654 (Supplementary Fig. S8A-8C). Upon realization that the gene annotation of 655 Capsaspora CDK1-3 was incomplete, we designed forward (5’- 656 GCTCAAGGAGGTCATCCACC-3’) and reverse (5’- 657 CTCTCTGCCCGATTACAAGC-3’) primers to PCR-amplify the unknown 658 sequence from both genomic DNA and cDNA (Supplementary Fig. S8A-8B). 659 Sanger sequencing of the amplified products revealed one more intron and one 660 more exon, which were used to reconstruct the missing sequence in the 661 assembly. We mapped the transcriptome of Replicate 2 – timepoint 9 against this 662 reconstructed sequence using tophat2[123] with standard parameters. 663 Identification of paired-end overlapping reads in the reconstructed exons using 664 Tablet [124] verified the results of Sanger sequencing (Supplementary Fig. S8A). 665 The updated cDNA and protein sequence of Capsaspora CDK1-3, which aligns 666 much better with human and yeast sequences (Supplementary Fig. S8C), can be 667 found in Supplementary File 6.

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668 Identification of orthologous genes

669 Groups of orthologs were generated using OrthoFinder v2.1.2 [99] using 670 default parameters (evalue 10e-3, MCL clustering) on a dataset of proteomes 671 found in Supplementary File 5[113,125–127]. 672 673 One-to-one orthologues were taken from a phylome of 6598 genes of 674 Capsaspora reconstructed in [61] using the algorithm by [128] 675 (http://phylomedb.org/phylome_100).

676 Determining cell cycle-regulated genes in H. sapiens, S. cerevisiae, 677 S. pombe and A. thaliana

678 We downloaded the lists of cell cycle regulated genes which can be found 679 at [32,41,43,44,50,91]. We translated the gene and probe names of their datasets 680 using BioMart[129] and Uniprot[113] tools (see Supplementary File 10).

681 Gene Age enrichment analysis

682 We used Count software[105] to assign the emergence of every orthogroup 683 –and therefore every Capsaspora gene- to a given ancestor in common between 684 species by Dollo Parsimony. We defined six different ages for Capsaspora: 0 685 “Capsaspora-specific”, 1 “Filozoa”, 2 “Holozoa”, 3 “Opisthokonta”, 4 “Unikonta”, 686 and 5 “Paneukaryotic”. All Capsaspora genes unassigned to any orthogroup were 687 defined as “Capsaspora-specific”. Gene age enrichment was calculated using 688 contingency tables and significance by Fisher exact test using R software ver. 689 3.4.4.[111], and was corrected for multi-test hypothesis using Bonferroni 690 correction.

691 Comparative analysis

692 Every periodic and non-periodic gene of each of the seven datasets were 693 assigned to their respective orthogroup, if any. We obtained seven lists of 694 orthogroups containing periodic genes of each species or cell type, and also 695 defined lists of orthogroups containing genes (regardless of periodic) of each of 696 the five species. A subset of periodic orthogroups (those containing at least one 697 periodic gene from at least one of the seven datasets) was generated, and plotted

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698 for periodicity, presence, or absence, in all the datasets using R gplots 699 library[112]. 700 701 Numbers and ratios of periodic shared orthogroups and one-to-one 702 orthologues, as well as binomial test p-values (see Fig.6D, Supplementary Fig. 703 S9, Supplementary File 11, Supplementary File 12), were calculated using R 704 software ver. 3.4.4. [127]. For each pair-wise comparison of species, e.g. 705 Capsaspora and Homo sapiens HeLa cells (Fig.6C), we took the number of 706 orthogroups they have in common as a total population, C. Then we looked at 707 the number of periodic orthogroups from each species that are within this total

708 population, p1 and p2, and calculated the null expectation (Aexp) as a product of 709 the ratios of these two subpopulations within the population of orthogroups in 710 common.

711 Reanalysis of cell cycle datasets using JTK_CYCLE and RAIN

712 We reanalyzed the datasets of gene expression of [44] (HU treatment 713 dataset), [91] (three replicates of elutriation), [43] (two replicates of wildtype 714 synchronous yeast cultures) , [41] (one replicate, thymidine block), [32] (four 715 replicates of double thymidine block), and [50] (a dataset of ~8000 genes 716 matching filtering criteria by the authors) using the same pipeline used in our 717 Capsaspora datasets. For those with replicates, periodicity ranks were calculated 718 for each replicate independently and summed at the end. 719 720 As every experiment comprised different numbers of cell cycles of different 721 length, we set up JTK and RAIN parameters to look for periodicity in time lapses 722 according to the author’s reports (see Supplementary File 10). We corrected for 723 the number of genes by setting a threshold of less than 10% of genes to be 724 periodic. Overlap between datasets of periodic genes was calculated using R 725 Software ver. 3.4.4. [111].

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1105 1106 Fig. 1: Experimental setup for cell cycle synchronization in Capsaspora. A: Effect 1107 of Hydroxyurea in Capsaspora cells. Control cells were treated with an equal volume of 1108 distilled water. HU treated cells were sampled every 2 hours, and a major decrease in 1109 2C cells is achieved after a minimum of 8 hours. B: Experimental layout used in this 1110 study. Two cell cultures growing independently for several generations were grown in 1111 fresh medium and kept in HU for a lapse of fifteen hours. After that, cells were released 1112 from HU and harvested every 45 minutes for 11 hours. Three different samples were 1113 taken from each culture at each time point. C: DNA content profile of synchronized 1114 Capsaspora cultures at representative time points of the whole experiment.

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1115 1116 Fig. 2: Cell division in Capsaspora. A: Time lapse of live imaging of a synchronous 1117 culture. Numbers indicate minutes since round up. White arrows indicate a cell dividing 1118 during the time lapse. Scale bar: 5µm. B: Fluorescence immunostaining of DNA (cyan) 1119 and Tubulin alpha (green) in Capsaspora synchronous cultures at different stages of cell 1120 division. White arrows indicate structures with a high concentration of tubulin. White 1121 dashed outline indicates cell perimeter. Scale bar: 5µm. C: Histogram depicting the 1122 number of cells at the moment of division in different times of the time lapse. Range goes 1123 from 7h to 11h. D: Stacked barplot showing the normalized, relative cell area for each 1124 daughter cell in a total of 101 events of cell division.

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1125 1126 Fig. 3: Detection of periodic genes in Capsaspora. A: Pearson correlation for the 1127 normalized expression level of every gene between replicates. Bright red indicates 1128 positive correlation, and dark purple indicates negative correlation. B: Spearman 1129 correlation between the two methods used to detect cell cycle regulated genes in 1130 Capsaspora. Scatter plots depicting the rank assigned for every gene by the 1131 JTK_CYCLE and RAIN on an average dataset of the two time-series replicates. These 1132 two algorithms rely on different approaches to finally assign, for every gene, a p-value 1133 interpreted as the probability that it can be considered periodic. For its proper functioning, 1134 we set the two algorithms to look for periodic behavior in a lapse of 11 to 13 hours, with 1135 time lapses of 0.75 hours. We assigned two ranks to every gene according to the p- 1136 values calculated by JTK_CYCLE and RAIN. Each dot represents a gene expressed in 1137 the time series. Colored dots represent the 801 genes that were finally taken as periodic 1138 according to our criteria. C: Pearson correlation for the top 801 cell cycle regulated 1139 genes, also based on normalized gene expression level. See Fig. 4 for more details. D:

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1140 Principal coordinate analysis on the set of 801 cell cycle regulated genes based on 1141 normalized gene expression level. Every dot represents a time point in the experiment.

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1142 1143 Fig. 4: The periodic transcriptional program of Capsaspora. A: heatmap of gene 1144 expression level depicting seven main clusters detected by Euclidean distance 1145 hierarchical clustering. Clusters were rearranged to visually represent their expression 1146 peaks over time. Black arrow and dividing cell indicate time of cell division (see Fig. 2). 1147 B: Top ten enriched gene ontology terms for every cluster shown in Fig. 4A. We 1148 considered an enrichment as significant when Bonferroni corrected p-value was lower 1149 than 0.05. For the full list of enriched GO terms, see Supplementary Table 4.1

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1150 1151 Fig. 5: Dynamics of the cyclin-CDK system and other cell cycle regulators in in 1152 Capsaspora. A: Normalized gene expression profile of the Cyclin A, B, and E genes 1153 found in Capsaspora. Normal and dashed lines indicate replicates 1 and 2, respectively. 1154 B: Normalized gene expression profile of the Capsaspora orthologue of CDK1/2/3 found 1155 by phylogenetic analyses (see Supplementary Fig. S7). Normal and dashed lines 1156 indicate replicates 1 and 2, respectively. C: Normalized gene expression level of 1157 Capsaspora orthologues of several G1/S regulators in animals. D: Normalized gene 1158 expression level of Capsaspora orthologues of several G2/M regulators in animals. 1159 Genes in C and D as described by [32,39,40,100]. A full list is depicted in Supplementary 1160 File 7.

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A All genes Periodic genes CapsasporaFilozoa HolozoaOpisthokontaUnikontaEukaryota 3.25% 1.12% 1.37% 2.06% 1.25% Filozoa Capsaspora 3.21% 2.5% 2.05% G1/S 23.9% **** *** * **** Holozoa Choanozoa (8 species) 17.6% S/G2 Opisthokonta Teretosporea (2 species) Unikonta G2/M Holomycota(7 species) *** ***** Eukaryota Amoebozoa(1 species) M 76.15% Other eukaryotes (9 species) 65.53% G1 **** *****

* p < 0.05 -20 20 p < 0.005 % enrichment **

B C U2OS cells p1 p2 HeLa cells H. sapiens Fibroblasts C

C. owczarzaki A S. cerevisiae p · p S. pombe A = 1 2 exp C A. thaliana Periodic orthogroups Absent Present Periodic Periodic orthogroups in common

D Budding Fission H. sapiens Arabidopsis yeast yeast HeLa U2OS Fibroblast Core human cell cycle 200 200 200 200 200 200 * 200

no. Periodic groups *** * 100 100 100 100 100 100 of orthologues shared * 100 *** * *

with Capsaspora 50 50 50 50 50 50 50 0 0 0 0 0 0 0

167/112 81/29 99/59 38/10 86/46 40/30 48/32 * 60 60 60 60 60 60 Observed number * no. Periodic one-to-one * 40 40 40 40 40 40 Expected number orthologues * 20 20 20 20 20 with Capsaspora 20 * p-value < 0.05 0 0 0 0 0 0 *** p-value < 10-9 1161 69/61 33/23 46/19 19/7 48/34 19/21 1162 Fig. 6: The Capsaspora periodic transcriptional program has more resemblances 1163 to that of animal cells. (Left) Tree depicting gene ages considered in this study. 1164 (Center) Gene age stratification profiles of the Capsaspora genome and the periodic 1165 transcriptional program. (Right) Gene age enrichment/depletion on each cluster of 1166 periodic genes in Capsaspora (see Fig. 4) compared to non-periodic genes. B: Heatmap 1167 showing periodic orthogroups in common in the tree of species used in the comparative. 1168 C: Venn diagrams indicating how the binomial tests were calculated for each pair-wise 1169 comparison of species, e.g. Capsaspora and Homo sapiens HeLa cells. White circles 1170 indicate orthogroups from each species. The intersection between these represents the 1171 orthogroups in common, C. p1 and p2 areas represent periodic genes of each species

1172 within C. Null expectation probability (Aexp) is calculated as the product of p1 and p2

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1173 divided by C. P-values of all the binomial tests are provided in Supplementary File 9 and 1174 Supplementary File 11. D: Bar plots indicating the amount of shared periodic 1175 orthogroups and/or periodic one-to-one orthologues between pairs of cell types or 1176 species. P-values of all the binomial tests are provided in Supplementary File 9. E: Gene 1177 age enrichment/depletion of Capsaspora periodic genes with a periodic co-orthologue in 1178 H. sapiens HeLa cells. Comparison was done against the rest of the Capsaspora periodic 1179 transcriptional program. Color code for bar plots as in Fig.6A.

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1180

Cln

cdc28

Clb Saccharomyces cerevisiae G1 S G2 M

CoCycE

CoCDK CoCycA 1/2/3 CoCycB Capsaspora owczarzaki G1 S G2 M

CDK6 CCND

CDK4 CCNE CDK2 CCNA Homo sapiens G1 S G2 M CDK1 CCNB

Cyclin levels Binding interaction

CDK levels Putative interaction 1181 1182 Fig. 7: a model of the dynamics of the Cyclin/CDK system in Capsaspora. Bold lines 1183 indicate cyclin levels and dashed lines indicate CDK levels across the cell cycle. In 1184 Capsaspora, several cell cycle cyclins may be binding to a single CDK that is not 1185 transcriptionally stable, an intermediate system that falls between yeast and human 1186 cyclin/CDKs. This suggests the expansions of cell cycle cyclins predate the expansion 1187 of cell cycle CDKs, which later acquired specific binding to cell cycle cyclins in animals. 1188

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A

RNA reads Kallisto (Bray et al., 2016)

Time-series transcriptome

average tpm > 1

Noise-removed trascriptome

JTK_CYCLE (Hughes et al., 2010) RAIN (Thaben and Westermark, 2014)

λ = 11.75 : 13.25 λ = 13.25 t = 0.75 t = 0.75

BH.Q p-val BH.Q p-val

JTK rank RAIN rank

Sum of ranks

B

Time-series transcriptome Time-series transcriptome Time-series transcriptome Time-series transcriptome (Replicate 1) (Replicate 2) (Replicate 1) (Replicate 2)

Time-series transcriptome Pipeline in Pipeline in (Average) A A

Pipeline in Top 2000 (loose Top 2000 (loose A cutof) cutof)

1774 genes (high score on indivi- dual replicates) Average genes with high score on indivi- dual replicates

Top 800 genes with high score on individual replicates + CAOG_04719 (Cyclin A) 1189 1190 Supplementary Fig. S1: Computational pipeline used to detect periodic genes in 1191 Capsaspora. A: Pipeline for ranking the transcripts on each experiment. RNA reads were 1192 processed using Kallisto, noise transcripts were filtered out, and JTK and RAIN were run 1193 in parallel with the indicated setup. Bonferroni-corrected p-values were ranked, and the 1194 sum of ranks was used as a final rank. B: Pipeline used to detect periodic transcripts in 1195 Capsaspora. Samples were treated separately to detect periodic transcripts with a loose

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1196 cutoff. This gene set was used to filter periodic genes ranked from an average dataset 1197 of the two replicates, out of which the top 800 (10% of the number of genes in 1198 Capsaspora) were selected.

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t1.1 t1.2 t2.2 t2.1 t3.1 t4.1 t3.2 t4.2 t5.1 t6.1 t5.2 t6.2 t7.2 t8.2 t9.2 t9.1 t8.1 t7.1 Unsynchronous t16.1 t15.1 t16.2 t15.2 t14.2 t11.1 t10.1 t12.1 t14.1 t13.1 t13.2 t12.2 t11.2 t10.2 t1.1 t1.2 t2.2 t2.1 t3.1 t4.1 t3.2 t4.2 t5.1 t6.1 t5.2 t6.2 t7.2 t8.2 t9.2 t9.1 t8.1 t7.1 -1 1 t16.1 t15.1 t16.2 t15.2 t14.2 t11.1 t10.1 t12.1 t14.1 t13.1 t13.2 t12.2 t11.2 t10.2 N= 8016 genes, all expressed in both replicates

1199 Replicate 1 Replicate 2 Unsynchronous 1200 Supplementary Fig. S2: clustering of time points of each replicate and the 1201 unsynchronized culture sampled as a control, based on a dissimilarity matrix of Pearson 1202 correlation. All the genes with an average expression level above 1 tpm in both replicates 1203 were used.

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1204 1205 Supplementary Fig. S3: A: Distributions of Pearson correlation between replicates of a 1206 set of randomly chosen 801 genes and the 801 genes defined as periodic. B: Fraction 1207 of variance explained by the different relative eigenvalues of the principal coordinate 1208 analysis (see Fig. 3D).

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1209 1210 Supplementary Fig. S4: A: Hierarchical clustering of the expression profiles of the 801 1211 periodic genes found in Capsaspora, and the color equivalences with the final clusters 1212 shown at Fig. 4A. B: Average expression level of Capsaspora periodic genes grouped 1213 by hierarchical clustering. C: t-SNE plot of all 801 genes in the periodic transcriptional 1214 program of Capsaspora, showing circle pattern as in Fig. 3D. Color code in both Fig.s 1215 follows the color code in Fig. 4. 1216

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1217 1218 Supplementary Fig. S5: K-means clustering of the periodic transcriptional program of 1219 Capsaspora. A: Average expression level of Capsaspora periodic genes grouped by k- 1220 means clustering. B: Top ten enriched GO terms of each cluster of periodic genes 1221 generated by k-means clustering. GO terms were considered significant when 1222 Bonferroni-corrected p-value was lower than 0.05. Full list available at Supplementary 1223 Fig. S3. C: Agreement between clustering methods. Heatmap showing the percentage 1224 of overlap between clusters by two methods. Overlap is calculated as the number of 1225 genes belonging to the same pair of clusters divided by the size of the smallest cluster 1226 in the pair. 1227

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1228 Fig. 4—source data 1: List of significant (Bonferroni p-value < 0.05) GeneOntology 1229 enrichments of each hierarchical cluster of periodic genes in Capsaspora. Available on 1230 Figshare: https://figshare.com/s/4d642c9854efe6d879a7

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anim-Pcau_10184 anim-Pcau_17439 anim-Pcau_8708 anim-Pcau_17582 anim100 -Pcau_2913 Fungi anim-Pcau_14740 95 anim-Skow_3477 100 81 anim-Skow_5667 97 100 CCNK-anim-Hsap_O75909 Embryophyta anim-Bfo_6439 100 anim-Dmel_596 96 anim-Nvec_20467 Early holomycota 100 anim-Tadh_7090 anim-Aque_27977 fungi-Scom_4685 90 99 Cyclin_N-fungi-Scer_1365 Metazoa fungi-Spom_1247 89 96 84 fungi-Mver_8118 hmycota-Patl_trans_11877 100 Early holozoa 97 hmycota-Patl_trans_5952 97 fungi-Spun_3267 85 hzoa-Clim_1863 hzoa-Sros_3301 Amoebozoa 81 99 hzoa-Mbre_6673 Cyclin K, hzoa-Mvib_trans_12052 100 96 hzoa-Pchi_trans_20614 Cyclin T1-2 clade SAR 70 hzoa-Mvib_trans_21293 hzoa-Cowc_CAOG_08557T0 88 hzoa-Cfra_3059 100 CCNK, CCNT1-2 Apusomonadida 71 hzoa-Sarc_g32273T hzoa-Cper_4412 70 anim-Pcau_20104 anim100 -Pcau_8726 97 anim-Pcau_8115 92 anim-Dmel_10298 anim-Aque_953 95 95 anim-Bfo_14332 CCNT1-anim-Hsap_O60563 94 100 CCNT2-anim-Hsap_O60583 92 100 anim-Nvec_11963 anim-Tadh_11487 90 anim-Mlei_6248 CYCT1_5-embryo-Atha_Q9FKE6 100 100 CYCT1_4-embryo-Atha_Q8GYM6 CYCT1_2-embryo-Atha_Q56YF8 59 84 100 CYCT1_3-embryo-Atha_Q8LBC0 93 CYCT1_1-embryo-Atha_Q9C8P7 apuso-Ttra_8083 100 85 SARhete-Esil_11951 amoe-Ddis_7531 35 apuso-Ttra_6745 fungi-Mver_1579 77 35 fungi-Scom_788 34 fungi-Spom_4905 99 fungi-Spun_584 48 fungi-Lbic_10837 SARhete-Esil_10089 anim-Bfo_9679 85 anim-Nvec_4007 77 CCNL1-anim-Hsap_Q9UK58 98 78 CCNL2-anim-Hsap_Q96S94 79 anim-Dmel_2592 anim-Pcau_4232 100 95 anim-Pcau_4868 100 anim-Skow_3479 71 anim-Tadh_5791 hzoa-Sros_976 99 hzoa-Mbre_6618 82 hzoa-Mvib_trans_38709 100 95 hzoa-Mvib_trans_49767 97 85 hzoa-Pchi_trans_32228 Cyclin L1-2 clade 93 hzoa-Cowc_CAOG_02877T0 hzoa-Cfra_3641 69 100 CCNL1-2 71 hzoa-Sarc_g9540T hzoa-Cper_12143 SARhete-Esil_13741 36 fungi-Spun_5170 73 fungi-Mver_10531 45 fungi-Lbic_8319 46 61 70 fungi-Scom_5254 hmycota-Patl_trans_3914 90 amoe-Ddis_6205 61 CYCL1_1-embryo-Atha_Q8RWV3 fungi-Spom_919 CCNQ-anim-Hsap_Q8N1B3 100 100 FAM58B-anim-Hsap_P0C7Q3 44 94 26 anim-Skow_5283 100 anim-Bfo_20007 95 anim-Dmel_5978 91 anim-Nvec_10584 25 anim-Tadh_10228 Cyclin Q clade SARhete-Esil_12621 84 98 hzoa-Cfra_1894 100 CCNQ 91 hzoa-Sarc_g25715T hzoa-Mbre_3589 95 hzoa-Sros_1985 99 99 SARhete-Esil_8561 94 hzoa-Clim_1404 hzoa-Sros_484 100 uncertain hzoa-Mbre_38 fungi-Scom_2199 100 80 fungi-Scom_6185 fungi-Lbic_11173 hzoa-Mvib_trans_1989 uncertain hzoa-Cfra_908 100 47 hzoa-Sarc_g8682T 47 hzoa-Pchi_trans_8355 45 hzoa-Clim_4075 hzoa-Cowc_CAOG_02974T0 50 16 hzoa-Cper_5244 fungi-Spom_4330 77 apuso-Ttra_9676 96 44 fungi-Scom_1767 96 99 SSN8-fungi-Scer_P47821 46 97 fungi-Lbic_9407 Cyclin C clade fungi-Mver_8137 100 fungi-Mver_9978 CCNC hmycota-Patl_trans_21537 anim-Bfo_24602 52 50 hzoa-Mbre_3568 74 CCNC-anim-Hsap_P24863 99 anim-Dmel_11558 92 anim-Pcau_3495 100 98 84 anim-Pcau_558 anim-Skow_20596 98 87 anim-Mlei_14265 99 anim-Nvec_11934 99 anim-Tadh_4967 anim-Aque_26103 100 CYCC1_2-embryo-Atha_Q9FJK7 100 87 CYCC1_1-embryo-Atha_Q9FJK6 amoe-Ddis_3877 hzoa-Mvib_trans_8119 uncertain anim-Pcau_249 anim100 -Pcau_9488 100 anim-Pcau_7814 63 anim-Dmel_7260 65 anim-Skow_19002 96 anim-Bfo_20801 100 64 anim-Bfo_4997 76 CCNH-anim-Hsap_P51946 anim-Tadh_253 48 98 82 anim-Mlei_5225 anim-Nvec_18286 91 Cyclin H clade anim-Aque_17934 hzoa-Cper_3117 87 hzoa-Mvib_trans_18901 100 CCNH hzoa-Mvib_trans_48332 92 83 hzoa-Pchi_trans_29357 100 30 hzoa-Pchi_trans_7105 apuso-Ttra_3216 66 hzoa-Cowc_CAOG_04347T0 93 hzoa-Cfra_6614 44 100 hzoa-Sarc_g10965T CYCH1_1-embryo-Atha_Q8W5S1 fungi-Scom_2754 48 44 41 fungi-Spom_1297 42 fungi-Mver_5196 fungi-Spun_1739 99 99 fungi-Lbic_384 57 62 98 CCL1-fungi-Scer_P37366 hmycota-Patl_trans_3913 100 hmycota-Patl_trans_9351 70 amoe-Ddis_9538 39 SARhete-Esil_170 hzoa-Sros_10367 64 hzoa-Mbre_2454 hzoa-Clim_4867 uncertain amoe-Ddis_2449 PCL7-fungi-Scer_P40186 100 100 PCL6-fungi-Scer_P40038 92 fungi-Lbic_10810 fungi-Spom_2015 77 fungi-Mver_2006 100 88 100 fungi-Mver_4133 fungi-Mver_5493 98 fungi-Mver_8131 fungi-Spun_3978 hzoa-Pchi_trans_1457 100 88 100 hzoa-Pchi_trans_14994 91 hzoa-Pvie_trans_38936 hzoa-Cowc_CAOG_00523T0 81 99 hzoa-Cper_5821 hzoa-Cfra_1061 100 98 hzoa-Sarc_g30947T 42 hzoa-Clim_5548 fungi-Spun_2307 34 75 fungi-Spun_3869 98 fungi-Mver_1795 fungi-Lbic_5229 93 PCL6,PCL7, CYCU1-4 clade 24 100 PHO80-fungi-Scer_P20052 32 hmycota-Patl_trans_1828 hzoa-Sros_6099 PCL6-PCL7 ( Saccharomyces cerevisiae ) amoe-Ddis_11658 100 CYCU1-4 ( Arabidopsis thaliana ) 93 amoe-Ddis_129 96 amoe-Ddis_5938 amoe-Ddis_610 CYCU2_2-embryo-Atha_Q9M205 100 CYCU2_1-embryo-Atha_Q9SHD3 100 CYCU1_1-embryo-Atha_Q9LJ45 28 100 99 CYCU3_1-embryo-Atha_Q8LB60 CYCU4_2-embryo-Atha_Q9LY16 100 100 100 CYCU4_3-embryo-Atha_Q9FKF6 CYCU4_1-embryo-Atha_O80513 66 Cyclin-embryo-Atha_26881 hzoa-Clim_1744 62 69 amoe-Ddis_5717 hzoa-Sros_2708 100 93 hzoa-Mbre_210 hzoa-Cper_12344 80 99 96 hmycota-Patl_trans_8218 96 SARhete-Esil_3591 74 hzoa-Pvie_trans_13289 73 hzoa-Cowc_CAOG_05449T0 hzoa-Cfra_1049 100 hzoa-Sarc_g18803T fungi-Scom_6395 hzoa-Cfra_2566 100 hzoa-Sarc_g25033T 100 hzoa-Cfra_8681 100 96 hzoa-Sarc_g3774T 99 hzoa-Sarc_g1729T hzoa-Cper_1446 93 hzoa-Mvib_trans_45724 100 100 hzoa-Pchi_trans_35328 hzoa-Cowc_CAOG_01396T0 79 hzoa-Cfra_1716 100 91 hzoa-Sarc_g5399T 97 amoe-Ddis_7029 99 78 83 fungi-Mver_1878 hzoa-Clim_812 hzoa-Sros_5204 100 85 hzoa-Mbre_2372 fungi-Spun_4725 100 hmycota-Patl_trans_495 82 94 SARhete-Esil_12046 97 SARhete-Esil_5957

68 93 anim-Tadh_9866 Y, Cyclin 88 95 anim-Mlei_5703 anim-Pcau_12408 92 CCNYL3-anim-Hsap_P0C7X3 100 99 CCNYL1-anim-Hsap_Q8N7R7 CCNY, CCNYL1-3 CCNYL2-anim-Hsap_Q5T2Q4 71 100 1-3 clade Y-like Cyclin 99 CCNY-anim-Hsap_Q8ND76 60 anim-Skow_12689 anim-Dmel_1573 100 anim-Bfo_15431 anim-Aque_9541 93 anim-Nvec_20837 amoe-Ddis_1551 fungi-Lbic_16142 100 fungi-Lbic_17040 100 fungi-Lbic_2129 100 94 fungi-Lbic_9970 hzoa-Pchi_trans_13202 100 59 hzoa-Pchi_trans_31274 hzoa-Pchi_trans_20808 100 hzoa-Pchi_trans_27966 100 93 hzoa-Pvie_trans_2778 100 hzoa-Pvie_trans_37094 88 PCL5-fungi-Scer_P38794 fungi-Mver_10179 100 95 hmycota-Patl_trans_11966 apuso-Ttra_4601 fungi-Spun_1110 100 98 hmycota-Patl_trans_20952 66 100 fungi-Mver_6067 99 fungi-Spun_3836 fungi-Spun_1518 100 97 fungi-Spun_407 fungi-Spun_8963 86 fungi-Lbic_1198 99 fungi-Scom_2850 99 64 PCL5,CLG1 clade fungi-Spun_6190 87 fungi-Spom_2799 ( 99 CLG1-fungi-Scer_P35190 ) fungi-Mver_12217 PCL5, CLG1 95 100 92 fungi-Mver_3510 Saccharomyces 92 fungi-Spom_993 81 fungi-Scom_6327 cerevisiae 100 fungi-Spun_8128 86 fungi-Spun_6106 94 apuso-Ttra_1555 91 hzoa-Cper_10793 hzoa-Cfra_2484 100 49 hzoa-Sarc_g9853T 99 hzoa-Cfra_2167 hzoa-Cfra_36 100 68 hzoa-Cfra_4130 hzoa-Cper_11136 100 96 hzoa-Cper_6413 hzoa-Cowc_CAOG_05778T0 100 68 hzoa-Mvib_trans_42382 hzoa-Clim_7233 PCL9-fungi-Scer_Q12477 97 100 100 PCL2-fungi-Scer_P25693 100 PCL1-fungi-Scer_P24867 fungi-Scom_3471 100 90 fungi-Scom_576 90 fungi-Lbic_16288 PCL1,PCL2,PCL9 clade fungi-Mver_348 100 96 98 fungi-Mver_9073 100 fungi-Spun_9204 42 fungi-Lbic_15406 PCL1, PCL2, PCL9 hmycota-Patl_trans_13164 ( 99 hzoa-Pchi_trans_8438 100 100 hzoa-Pvie_trans_25365 Saccharomyces cerevisiae ) 98 94 hzoa-Cper_5871 hzoa-Cfra_6271 100 99 hzoa-Sarc_g9741T fungi-Spun_8185 anim-Aque_27969 87 87 anim-Nvec_25591 87 anim-Bfo_860 98 96 anim-Pcau_19037 98 100 Cyclin-anim-Hsap_4632 86 anim-Skow_17744 100 anim-Dmel_12179 anim-Tadh_610 uncertain hzoa-Cfra_6142 95 100 100 hzoa-Sarc_g3868T 100 hzoa-Cfra_8333 hzoa-Cfra_3077 100 hzoa-Sarc_g33089T hzoa-Cper_4071 amoe-Ddis_6701 PCL8-fungi-Scer_Q08966 100 PCL10-fungi-Scer_P53124 CYCB2_3-embryo-Atha_Q9LDM4 100 100 CYCB2_5-embryo-Atha_Q9LM91 100 CYCB2_4-embryo-Atha_Q9SFW6 CYCB2_2-embryo-Atha_Q39070 100 35 CYCB2_1-embryo-Atha_Q39068 CYCB3_1-embryo-Atha_Q9SA32 CYCB1_5-embryo-Atha_Q39072 45 100 100 CYCB1_2-embryo-Atha_Q39067 100 CYCB1_3-embryo-Atha_Q39069 86 100 CYCB1_1-embryo-Atha_P30183 CYCB1_4-embryo-Atha_O48790 SARhete-Esil_13892 45 42 SARhete-Esil_8106 amoe-Ddis_10917 93 CCNO-anim-Hsap_P22674 84 anim-Skow_12887 100 100 anim-Skow_748 94 anim-Skow_8803 89 66 CNTD2-anim-Hsap_Q9H8S5 anim-Mlei_6149 hzoa-Clim_2350 96 87 hzoa-Cper_9549 amoe-Ddis_7906 anim-Pcau_2268 100 28 anim-Skow_17328 anim-Aque_19485 94 CCNB1-anim-Hsap_P14635 100 93 100 CCNB2-anim-Hsap_O95067 79 anim-Nvec_842 hzoa-Sros_6136 100 hzoa-Mbre_450 hzoa-Mvib_trans_22975 75 100 100 hzoa-Mvib_trans_46303 71 hzoa-Mvib_trans_21780 95 hzoa-Cowc_CAOG_07647T0 hzoa-Mvib_trans_44055 68 100 71 67 hzoa-Mvib_trans_47701 hzoa-Pchi_trans_18829 96 hzoa-Pchi_trans_29339 hzoa-Cfra_4864 100 34 78 hzoa-Sarc_g32274T anim-Mlei_3102 anim-Tadh_4908 Cyclin O, CNTD2, 84 83 68 anim-Nvec_17647 anim-Dmel_7912 Cyclin B1-3 clade apuso-Ttra_1220 anim-Aque_2852 CCNO, CNTD2, CCNB1-3 hmycota-Patl_trans_21245 100 100 hmycota-Patl_trans_8159 98 fungi-Spom_1847 98 fungi-Mver_12592 99 fungi-Spom_2745 99 fungi-Scom_3073 CLB3-fungi-Scer_P24870 100 74 46 CLB4-fungi-Scer_P24871 fungi-Lbic_8233 13 CLB5-fungi-Scer_P30283 100 CLB6-fungi-Scer_P32943 36 CLB1-fungi-Scer_P24868 100 26 CLB2-fungi-Scer_P24869 fungi-Spun_3924 79 68 fungi-Mver_10187 37 94 fungi-Scom_2430 fungi-Spom_3324 80 98 fungi-Spom_4195 fungi-Lbic_2613 76 fungi-Mver_1118 anim-Pcau_11740 anim-Pcau_18438 anim98 -Pcau_3663 1an00im-Pcau_17982 100 anim-Pcau_18853 94 anim-Dmel_8465 anim-Skow_15769 89 anim-Aque_19741 43 95 91 CCNB3-anim-Hsap_Q8WWL7 100 anim-Nvec_3187 anim-Tadh_11269 100 88 anim-Mlei_15272 hzoa-Sros_121 99 hzoa-Pchi_trans_17940 hmycota-Patl_trans_17116 100 78 hmycota-Patl_trans_6739 62 SARhete-Esil_12631 fungi-Spom_1789 CCNA2-anim-Hsap_P20248 100 89 CCNA1-anim-Hsap_P78396 60 anim-Pcau_7855 59 anim-Bfo_16938 anim-Dmel_6910 76 anim-Skow_10626 an100im-Skow_9563 94 anim-Skow_2078 anim-Tadh_1231 49 94 anim-Nvec_10488 anim-Mlei_11718 100 95 anim-Mlei_9801 67 anim-Aque_2179 hzoa-Sros_7590 46 100 11 63 hzoa-Mbre_8968 hzoa-Cper_4246 89 hzoa-Cowc_CAOG_04719T0 hzoa-Pchi_trans_21304 Cyclin A1-2 clade 100 46 hzoa-Pchi_trans_33112 CCNA1-2 hmycota-Patl_trans_14122 100 hmycota-Patl_trans_857 CYCA3_3-embryo-Atha_A0MEB5 100 100 CYCA3_2-embryo-Atha_Q9C6A9 69 100 CYCA3_4-embryo-Atha_Q3ECW2 CYCA3_1-embryo-Atha_Q9FMH5 61 CYCA2_3-embryo-Atha_Q38819 100 CYCA2_4-embryo-Atha_Q9C968 100 CYCA2_1-embryo-Atha_Q39071 100 100 CYCA2_2-embryo-Atha_Q147G5 CYCA1_2-embryo-Atha_Q9FVX0 73 100 CYCA1_1-embryo-Atha_Q9C6Y3 apuso-Ttra_3533 14 anim-Pcau_16886 53 100 100 anim-Pcau_2381 96 anim-Bfo_24793 97 anim-Skow_20640 CCNF-anim-Hsap_P41002 88 hzoa-Sros_4758 100 Cyclin F clade 93 98 hzoa-Mbre_3994 anim-Tadh_5123 6 CCNF 99 anim-Nvec_2334 100 anim-Aque_18045 81 hzoa-Cper_8533 SARhete-Esil_2470 anim-Tadh_2050 24 24 hzoa-Cowc_CAOG_01199T0 uncertain 12 hzoa-Mvib_trans_35650 anim-Mlei_2028 75 hzoa-Mvib_trans_27656 5 apuso-Ttra_1296 anim-Skow_18898 anim-Skow_618 anim-Skow_20578100 anim-Skow_19949 95 anim-Aque_13227 97 94 97 CCNE1-anim-Hsap_P24864 CCNE2-anim-Hsap_O96020 53 anim-Bfo_4296 anim-Pcau_15762 100 Cyclin E1-2 clade 73 29 55 anim-Pcau_4140 anim-Nvec_15174 CCNE1-2 66 anim-Dmel_691 100 anim-Tadh_4244 anim-Mlei_2041 92 apuso-Ttra_3151 72 hmycota-Patl_trans_16468 93 22 99 hzoa-Cfra_8417 100 hzoa-Sarc_g2320T hzoa-Cowc_CAOG_01951T0 hzoa-Clim_6588 uncertain anim-Aque_26180 87 44 fungi-Mver_1506 12 CCSDS-embryo-Atha_Q1PFW3 uncertain hzoa-Cfra_3473 51 fungi-Spun_8405 SARhete-Esil_11685 99 99 CCNJL-anim-Hsap_Q8IV13 83 CCNJ-anim-Hsap_Q5T5M9 97 anim-Pcau_17252 anim-Bfo_18401 20 81 98 anim-Skow_6299 98 anim-Mlei_8267 98 anim-Nvec_1186 Cyclin J, Cyclin J-like clade 98 anim-Aque_10269 87 hzoa-Clim_259 100 anim-Dmel_13631 CCNJ, CCNJL 75 hzoa-Mbre_663 87 amoe-Ddis_452 68 hzoa-Cper_7391 hzoa-Clim_977 fungi-Lbic_9136 100 45 fungi-Scom_2576 88 fungi-Mver_7875 25 99 fungi-Spun_1804 uncertain 90 fungi-Spom_1195 CLN1-fungi-Scer_P20437 100 93 CLN2-fungi-Scer_P20438 95 CLN3-fungi-Scer_P13365 80 fungi-Spun_2832 hzoa-Mbre_273 anim-Dmel_7629 85 85 anim-Skow_14005 90 anim-Bfo_8893 CCNG2-anim-Hsap_Q16589 100 97 CCNG1-anim-Hsap_P51959 93 anim-Nvec_25975 anim-Nvec_22648 95 CCNI2-anim-Hsap_Q6ZMN8 94 94 CCNI-anim-Hsap_Q14094 100 91 anim-Bfo_5216 anim-Skow_9076 anim-Mlei_1921 100 hzoa-Pchi_trans_1630 100 Cyclin G1-2 10hz0 oa-Pvie_trans_40884 100 hzoa-Pchi_trans_22623 100 hzoa-Cowc_CAOG_00179T0 Cyclin I1-2 clade hzoa-Cper_4341 100 hzoa-Cfra_7211 CCNG1-2, CCNI1-2 100 43 hzoa-Sarc_g19862T hzoa-Cper_8772 58 hzoa-Pchi_trans_37751 100 31 hzoa-Pvie_trans_44916 100 hzoa-Cowc_CAOG_03927T0 hzoa-Mvib_trans_17749 hz100oa-Mvib_trans_26758 hzoa-Mvib_trans_24591 anim-Nvec_21509 62 CCND1-anim-Hsap_P24385 61 CCND3-anim-Hsap_P30281 94 100 95 62 CCND2-anim-Hsap_P30279 anim-Bfo_24067 91 97 92 anim-Tadh_5426 Cyclin D1-3clade anim-Skow_1747 100 anim-Pcau_6543 CCND1-3 100 anim-Dmel_9475 98 anim-Mlei_9590 hzoa-Clim_4785 fungi-Spun_8469 28 82 98 anim-Mlei_10351 hmycota-Patl_trans_14630

53 hzoa-Mvib_trans_31170 D1-3 clade I1-2, Cyclin G1-2, Cyclin Cyclin 19 64 100 100 hzoa-Mvib_trans_46459 hzoa-Mvib_trans_47563 hzoa-Pchi_trans_16621 100 63 hzoa-Pvie_trans_26548 hzoa-Cper_6347 CYCD3_2-embryo-Atha_Q9FGQ7 100 100 CYCD3_3-embryo-Atha_Q9SN11 96 CYCD3_1-embryo-Atha_P42753 16 91 CYCD7_1-embryo-Atha_Q9LZM0 90 CYCD5_1-embryo-Atha_Q2V3B2 18 CYCD1_1-embryo-Atha_P42751 CYCD6_1-embryo-Atha_Q9ZR04 100 CYCD4_2-embryo-Atha_Q0WQN9 100 100 CYCD4_1-embryo-Atha_Q8LGA1 CYCD2_1-embryo-Atha_P42752 SARhete-Esil_1443 96 SARhete-Esil_6293 anim-Bfo_1392 100 anim-Bfo_3818 100 CABLES1-anim-Hsap_Q8TDN4 100 68 97 CABLES2-anim-Hsap_Q9BTV7 anim-Dmel_8983 100 97 anim-Pcau_1708 17 anim-Tadh_2547 13 100 anim-Nvec_8569 anim-Aque_7147 76 anim-Mlei_5118 62 hzoa-Cfra_8240 100 79 hzoa-Sarc_g4871T CDK5 and ABL1 enzyme 67 SARhete-Esil_16223 23 hzoa-Sros_4450 100 hzoa-Mbre_8694 substrate (CABLES)1-2 clade hzoa-Mvib_trans_12053 7 100 CABLES1-2 94 78 hzoa-Mvib_trans_37879 hzoa-Cper_6968 apuso-Ttra_3627 96 99 23 amoe-Ddis_3624 hzoa-Clim_5605 100 fungi-Mver_7389 93 9 hmycota-Patl_trans_11300 97 fungi-Spun_6990 SARhete-Esil_13595 hzoa-Cper_2707 hzoa-Cfra_5044 100 uncertain 4 hzoa-Sarc_g10721T hzoa-Sros_3764 anim-Tadh_720 95 37 CNTD1-anim-Hsap_Q8N815 92 anim-Mlei_14432 anim-Bfo_14176 Cyclin N-terminal domain containing protein (CNTD) 1 clade 98 100 anim-Skow_9598 anim-Aque_13523 100 CNTD1 99 99 anim-Pcau_5287 hzoa-Cowc_CAOG_00166T0 CYCJ18-embryo-Atha_Q9C5X2 1231 0.5

56 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1232 Supplementary Fig. S6: Unrooted maximum likelihood phylogenetic tree (IQ-TREE) 1233 inferred from cyclins sequences of 30 eukaryotic species (see methods). Nodal support 1234 values (1000- bootstrap replicates by UFBoot) are shown in all nodes. Eukaryotic 1235 sequence names are abbreviated with the four-letter code (see methods) and colored 1236 according to their major taxonomic group (see panel).

57 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

CDK13-anim-Hsap_Q14004 100 96 CDK12-anim-Hsap_Q9NYV4 anim-Bfo_24817 98 anim-Pcau_13303 100 100 99 anim-Pcau_4388 anim-Dmel_7310 100 anim-Skow_5575 anim-Nvec_15718 97 100 anim-Nvec_18842 81 anim-Tadh_7043 96 hzoa-Mbre_8190 97 anim-Aque_4924 CDK12-13 clade 71 anim-Mlei_9290 hzoa-Cfra_4367 100 hzoa-Sarc_g31835T 67 hzoa-Clim_1184 47 hzoa-Pvie_trans_6845 92 99 hzoa-Cper_9396 80 47 hzoa-Mvib_trans_7836 hzoa-Cowc_CAOG_02335T0 57 hzoa-Sros_4425 hzoa-Sros_9312 99 hzoa-Mbre_8926 95 hzoa-Sros_9376 CDK9, CDK12-13 clade 99 hzoa-Mbre_399 anim-Skow_11872 100 99 anim-Skow_16585 99 anim-Nvec_25273 Metazoa anim-Bfo_22962 99 100 CDK9-anim-Hsap_P50750 anim-Dmel_12945 100 100 Early holozoa 100 anim-Pcau_18794 97 anim-Tadh_3035 100 anim-Mlei_14352 anim-Aque_12233 1a0n0im-Aque_8291 39 hzoa-Cfra_128 CDK9 clade 100 hzoa-Sarc_g32282T 100 hzoa-Cfra_2820 67 70 100 hzoa-Sarc_g9622T hzoa-Clim_3431 87 hzoa-Cper_2392 hzoa-Mvib_trans_2878 100 94 91 hzoa-Mvib_trans_51583 hzoa-Cowc_CAOG_07450T0 hzoa-Pchi_trans_22663 anim-Pcau_12331 anim-Pcau_15854 anim-Pcau_3641 anim-Pcau_15175 anim-Pcau_3904 an96 im-Pcau_15005 10an0im-Pcau_12672 44 anim-Pcau_1234 98 anim-Dmel_8500 anim-Bfo_19731 100 72 97 anim-Bfo_23080 58 anim-Bfo_20770 anim-Skow_3765 9038 CDK11-anim-Hsap_Q9UQ88 100 81 CDK11B-anim-Hsap_P21127 94 anim-Mlei_8608 CDK11, CDK11B clade anim-Nvec_19275 99 anim-Tadh_5171 99 anim-Aque_21363 hzoa-Sarc_g11469T 100 98 100 hzoa-Sarc_g16403T hzoa-Cfra_643 97 hzoa-Cper_7910 hzoa-Sros_3835 96 100 94 hzoa-Mbre_2541 hzoa-Pchi_trans_19670 hzoa-Cowc_CAOG_08444T0 99 hzoa-Mvib_trans_12945 anim-Skow_14813 100 97 CDK10-anim-Hsap_Q15131 100 anim-Pcau_18489 100 100 anim-Bfo_1457 100 anim-Dmel_7802 100 anim-Nvec_10309 87 anim-Tadh_8947 CDK10 clade anim-Aque_29674 90 87 hzoa-Mvib_trans_18739 hzoa-Sros_10860 100 100 hzoa-Mbre_4176 hzoa-Cfra_5749 100 hzoa-Sarc_g11690T anim-Tadh_2131 85 56 CDK7-anim-Hsap_P50613 44 anim-Dmel_1 46 anim-Skow_5676 anim-Nvec_21519 8841 anim-Pcau_523 64 58 anim-Bfo_23618 88 anim-Aque_2556 anim-Mlei_3908 hzoa-Pchi_trans_14731 93 81 h10z0oa-Pchi_trans_24448 CDK7 clade 89 hzoa-Pchi_trans_15675 hzoa-Sros_1456 89 100 hzoa-Mbre_7331 72 76 hzoa-Cowc_CAOG_01914T0 hzoa-Cper_10723 hzoa-Cfra_926 100 100 100 hzoa-Sarc_g20222T 92 hzoa-Sarc_g14028T hzoa-Clim_2222 hzoa-Mvib_trans_33862 anim-Pcau_15273 anim-Pcau_17113 anim-Pcau_9934 10an0im-Pcau_18737 39 anim-Pcau_15614 CDK20-anim-Hsap_Q8IZL9 99 41 anim-Bfo_10737 25 74 8 anim-Skow_8676 anim-Nvec_13356 91 anim-Aque_8646 anim-Tadh_9097 CDK20 clade 81 hzoa-Cper_3557 81 94 hzoa-Pchi_trans_25058 10hz0oa-Pchi_trans_26250 hzoa-Sros_11004 18 100 98 hzoa-Mbre_7075 anim-Mlei_15971 32 hzoa-Clim_6855 93 anim-Dmel_8629 CDK19-anim-Hsap_Q9BWU1 100 76CDK8-anim-Hsap_P49336 68 anim-Dmel_13375 anim-Bfo_20101 86 anim100 -Bfo_357 81 anim-Pcau_597 42 anim-Skow_20135 anim-Tadh_1369 94 100 94 anim-Tadh_1516 93 anim-Mlei_15109 100 anim-Nvec_10438 100 68 anim-Nvec_2410 anim-Aque_2703 CDK8, CDK19 clade 52 hzoa-Pchi_trans_34417 60 hzoa-Clim_4259 93 hzoa-Cper_1395 96 hzoa-Cowc_CAOG_02016T0 hzoa-Cfra_2861 100 91 hzoa-Sarc_g15890T hzoa-Mvib_trans_13540 76 100 1hz00oa-Mvib_trans_43971 hzoa-Mvib_trans_18967 100 hzoa-Sros_10309 100 hzoa-Mbre_2549 hzoa-Cowc_CAOG_07268T0 70 hzoa-Pvie_trans_17197 100 99 hzoa-Pvie_trans_7774 78 hzoa-Sarc_g19171T hzoa-Clim_40 hzoa-Cper_8357 uncertain 49 anim-Mlei_10274 hzoa-Cfra_2462 100 hzoa-Sarc_g2710T 100 hzoa-Cfra_7536 100 100hzoa-Sarc_g28924T 100 hzoa-Clim_2352 hzoa-Cper_1177 99 hzoa-Mvib_trans_43823 100 100 hzoa-Mvib_trans_50759 72 100 hzoa-Pvie_trans_10208 hzoa-Cowc_CAOG_01997T0 hzoa-Sros_7576 100 hzoa-Mbre_5892 anim-Nvec_4938 70 CDK17-anim-Hsap_Q00537 74 66 anim-Skow_9698 6968 CDK16-anim-Hsap_Q00536 53 CDK18-anim-Hsap_Q07002 99 anim-Bfo_15057 CDK14-18 clade 85 anim-Tadh_9880 100 anim-Aque_19214 anim-Mlei_5461 99 CDK14-anim-Hsap_O94921 100 89 CDK15-anim-Hsap_Q96Q40 85 anim-Skow_16325 68 anim-Pcau_2822 50 anim-Dmel_9547 99 99 anim-Tadh_2703 99 anim-Nvec_73 100 anim-Aque_25005 31 anim-Mlei_2369 hzoa-Cowc_CAOG_07047T0 10h0zoa-Cowc_CAOG_07047T1 CDK5, CDK14-18 clade 82 anim-Pcau_11556 19 91 anim-Skow_11130 87 anim-Bfo_6119 88 CDK5-anim-Hsap_Q00535 anim-Dmel_4599 CDK5 clade 79 100 87 anim-Nvec_4817 100 anim-Aque_20336 98 anim-Tadh_9803 anim-Mlei_7060 hzoa-Mvib_trans_40268 100 77 hzoa-Mvib_trans_48779 hzoa-Pchi_trans_24562 100 100 hzoa-Pvie_trans_15360 98 hzoa-Cowc_CAOG_00023T0 CDK5, CDK14-18 clade 98 97 hzoa-Cper_7668 hzoa-Cfra_4126 98 100hzoa-Sarc_g26275T 25 hzoa-Clim_5704 hzoa-Sros_4078 100 hzoa-Mbre_5478 uncertain hzoa-Clim_7435 anim-Pcau_14769 100 90 anim-Pcau_7326 83 anim-Dmel_7079 84 anim-Bfo_5482 97 anim-Skow_9915 CDK1-anim-Hsap_P06493 anim-Nvec_15549 95 100 86 anim-Nvec_26208 CDK1 clade 100 79 anim-Aque_15761 anim-Tadh_4841 63 100 anim-Mlei_3311 hzoa-Cowc_CAOG_07905T0 96 hzoa-Mvib_trans_252 100 62 hzoa-Pchi_trans_9915 10h0zoa-Pvie_trans_43642 CDK1-3 clade hzoa-Cfra_3438 100 96 hzoa-Sarc_g28324T CDK1-3 clade hzoa-Cper_6385 anim-Dmel_5365 59 80 80 CDK3-anim-Hsap_Q00526 67 20 CDK2-anim-Hsap_P24941 80 anim-Pcau_18839 78 anim-Skow_8589 97 anim-Nvec_17803 CDK2-3 clade 92 100 anim-Tadh_11080 anim-Aque_8227 98 62 anim-Mlei_13221 hzoa-Sros_1426 100 96 hzoa-Mbre_2443 CDK1-3 clade hzoa-Sros_8415 hzoa-Sarc_g29060T hzoa-Cfra_4521 uncertain 100 82 hzoa-Sarc_g2703T 21 69 hzoa-Clim_480 anim-Bfo_14423 100 anim-Pcau_4175 100 CDK4-anim-Hsap_P11802 95 100 CDK6-anim-Hsap_Q00534 57 97 anim-Skow_7954 anim-Tadh_7198 CDK4, CDK6 clade 97 87 anim-Nvec_26627 99 anim-Dmel_5125 anim-Mlei_4551 hzoa-Sros_7014 86 hzoa-Mbre_7459 uncertain anim-Pcau_15496 anim-Pcau_5114 an100im-Pcau_20544 100 anim-Pcau_19343 CDKL1-anim-Hsap_Q00532 anim-Skow_22043 80 100 95 anim-Skow_4058 11 78 anim-Dmel_6401 anim-Bfo_3805 12 CDKL4-anim-Hsap_Q5MAI5 97 anim-Tadh_10021 CDKL1, CDKL4 clade 98 anim-Mlei_3638 anim-Nvec_10281 hzoa-Pchi_trans_29500 97 hz100oa-Pchi_trans_4658 10hzoa-0 Pvie_trans_2276 100 10hzoa-0 Pvie_trans_4175 100 hzoa-Cper_1142 hzoa-Sros_7003 100 hzoa-Mbre_224 anim-Pcau_12817 97 100 anim-Pcau_18059 1anim00 -Pcau_4774 92 anim-Pcau_15367 CDKL2-anim-Hsap_Q92772 anim-Aque_7226 83 100 CDKL2-3 clade 100 anim-Skow_16171 98 98 anim-Nvec_20676 anim-Bfo_26813 anim-Mlei_14211 83 CDKL3-anim-Hsap_Q8IVW4 97 anim-Nvec_23747 98 anim-Skow_6014 92 anim-Pcau_4753 94 98 CDKL5-anim-Hsap_O76039 anim-Bfo_27840 100 anim-Bfo_3942 100 hzoa-Pchi_trans_28329 80 CDKL5 clade h10z0oa-Pchi_trans_3343 93 100 96 hzoa-Pchi_trans_4920 hzoa-Cper_1507 100 anim-Nvec_21228 87 hzoa-Sros_3898 100 hzoa-Mbre_5961 CDKL1-5 clade hzoa-Cper_10301 56 anim-Tadh_9758 93 87 anim-Mlei_9684 hzoa-Clim_6714 anim-Aque_18770 18 18 hzoa-Cper_10164 hzoa-Cper_2212 uncertain 16 hzoa-Sros_4949 96 73 hzoa-Mbre_2808 hzoa-Mvib_trans_46313 anim-Mlei_13267 38 hzoa-Mvib_trans_35035 outgroup-anim-Hsap_18941 100outgroup-anim-Hsap_486 outgroup 1237 0.5

58 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1238 Supplementary Fig. S7: Maximum likelihood phylogenetic tree (IQ-TREE) inferred from 1239 CDK sequences of early-branching holozoan species and animals (see methods). 1240 Statistical support values (1000-replicates UFBoot) are shown in all nodes. Eukaryotic 1241 sequence names are abbreviated with the four-letter code (see methods) and colored 1242 according to their major taxonomic group (see panel).

59 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A

CAOG_07905 Forward *** supercont4.16: 417,379 NNNNNNNNNNNNNNNNNNNNNNNNN

Reverse

Sanger sequencing Exon & alignment CDK1 conserved *** translated sequence CAOG_07905 *** *** Primer supercont4.16: 417,379

tophat2 (Cell cycle transcriptome R2.T9)

CAOG_07905

supercont4.16: 417,379

Pair-end overlaps

B C

gDNA amplifcations cDNA amplifcations

H. sapiens Capsaspora S. cerevisiae

H. sapiens Capsaspora 1500 S. cerevisiae

1000 H. sapiens Capsaspora S. cerevisiae 500 400 H. sapiens Capsaspora S. cerevisiae 200 100 H. sapiens Capsaspora S. cerevisiae 1243 1244 Supplementary Fig. S8. A: Schematic representation of the genomic locus of 1245 Capsaspora CDK1-3 gene showing exons, splicing sites, non-annotated regions of 1246 predicted sequence, and mapping of mRNA reads. B: PCR amplifications of Capsaspora 1247 CDK1 using primers detailed in Methods and A, using genomic DNA and cDNA as 1248 templates. Arrows indicate size of the products sent for sequencing. C: Alignment of H. 1249 sapiens and S. cerevisiae CDK1 genes, and the Capsaspora updated CDK1-3 1250 sequence, using Geneious v8.1.9.

60 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

A Correlation between methods in other datasets

A. thaliana (Menges, 2004)aa S. cerevisiae (Orlando, 2008) S. pombe (Rustici, 2004)

Original Our method Original Our method Original Our method publication publication publication 805 76 873 480 280 419 165 222 238

H. sapiens HeLa cells (Dominguez, 2016) H. sapiens fbroblasts (Bar-Joseph, 2016) H. sapiens U2OS cells (Grant, 2013)

Original Our method Original Our method Original Our method publication publication publication

899 893 915 269 191 1470 641 604 736

B Budding Fission H. sapiens Arabidopsis yeast yeast HeLa U2OS Fibroblast Core human cell cycle 200 200 200 200 200 200 * 200 * no. Periodic groups * of orthologues shared * * * 100 100 100 100 100 100 100 with Capsaspora 50 50 50 50 50 50 *** 50 0 0 0 0 0 0 0 171/118.358 132/94.343 111/69.009 31/8.181 90/59.624 69/44.278 83/47.492

Observed number 60 60 60 60 60 60 no. Periodic one-to-one Expected number 40 40 40 40 40 orthologues 40 * * p-value < 0.05 20 20 20 20 20 with Capsaspora 20 0 0 0 0 0 0 *** p-value < 1,00-9 1251 78/68 38/29.721 53/48.394 15/5 48/56 19/21 1252 Supplementary Fig. S9: A Reanalysis of previous cell cycle datasets in model 1253 organisms. A: Scatter plots of ranks by JTK and RAIN for each dataset of each species 1254 used in the comparative analysis (see Fig. 6.D and Results section). Datasets were 1255 processed as indicated in Supplementary File 10, and Material and methods. Depending 1256 on the dataset, we could recover between one third and more than half of the originally 1257 described periodic genes, except Arabidopsis where the agreement was very low. B: 1258 Bar plots indicating the amount of shared periodic orthogroups and/or periodic one-to-

61 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1259 one orthologues between pairs of cell types or species, using our own lists of periodic 1260 genes. P-values of all the binomial tests are provided in Supplementary File 9.

62 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1261 Video 1: Synchronized cells of Capsaspora undergoing cell division. Time interval 1262 between frames is 1 minute. The movie is played at 7fps. Scale bar = 5µm. Available on 1263 Figshare: https://figshare.com/s/4d642c9854efe6d879a7 1264 1265 Supplementary File 1: Tables of transcript per million of each replicate. Available on 1266 Figshare: https://figshare.com/s/4d642c9854efe6d879a7 1267 1268 Supplementary File 2: List of periodic genes in Capsaspora, containing information for 1269 each gene about cluster membership, gene age, and orthologs in other species. 1270 Available on Figshare: https://figshare.com/s/4d642c9854efe6d879a7 1271 1272 Supplementary File 3: Gene Ontology enrichments for all the clusters in the periodic 1273 transcriptional program of Capsaspora. Available on Figshare: 1274 https://figshare.com/s/4d642c9854efe6d879a7 1275 1276 Supplementary File 4: FASTA formatted sequences of cyclins and CDKs used in the 1277 phylogenetic analyses (see Supplementary Fig. S6 and Supplementary Fig. S7). 1278 Available on Figshare: https://figshare.com/s/4d642c9854efe6d879a7 1279 1280 Supplementary File 5: List of species used in the phylogenetic analyses and in the 1281 generation of groups of orthologues. Available on Figshare: 1282 https://figshare.com/s/4d642c9854efe6d879a7 1283 1284 Supplementary File 6: FASTA formatted sequences of newly annotated Capsaspora 1285 CDK1-2-3 CDS and protein translation. Available on Figshare: 1286 https://figshare.com/s/4d642c9854efe6d879a7 1287 1288 Supplementary File 7: List of cell cycle regulators in humans (described in 1289 [32,39,40,100]), and their respective orthologs in Capsaspora defined by OrthoFinder 1290 and/or phylome data (see Results and Methods). Bold indicates genes that have been 1291 plotted in Fig. 5C or 5D. Available on Figshare: 1292 https://figshare.com/s/4d642c9854efe6d879a7 1293 1294 Supplementary File 8: List of cell cycle regulators in humans (described in 1295 [32,39,40,100]), that also have at least one periodic co-ortholog in Capsaspora, 1296 defined by OrthoFinder (see Results and Methods). Available on Figshare: 1297 https://figshare.com/s/4d642c9854efe6d879a7

63 bioRxiv preprint doi: https://doi.org/10.1101/719534; this version posted July 31, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC 4.0 International license.

1298 1299 Supplementary File 9: Metrics of gene age enrichment and depletion for the gene 1300 clusters of the periodic transcriptional program of Capsaspora, and their corresponding 1301 Fisher Test p-values. Available on Figshare: 1302 https://figshare.com/s/4d642c9854efe6d879a7 1303 1304 Supplementary File 10: Procedure used to retrieve identifiers for the datasets of H. 1305 sapiens, S. cerevisiae, S. pombe, and A. thaliana, and parameters used to set up 1306 JTK_CYCLE and RAIN in the reanalysis. Available on Figshare: 1307 https://figshare.com/s/4d642c9854efe6d879a7 1308 1309 Supplementary File 11: Metrics of shared periodic orthogroups (OG) and one-to-one 1310 orthologues between Capsaspora and the rest of cell types and species. Available on 1311 Figshare: https://figshare.com/s/4d642c9854efe6d879a7 1312 1313 Supplementary File 12: Metrics of shared periodic orthogroups (OG) for all 1314 comparisons between pairs of species, using periodic genes from the literature and 1315 using our own sets of periodic genes. Available on Figshare: 1316 https://figshare.com/s/4d642c9854efe6d879a7

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