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Proc Natl Acad Sci U S A
Proc Natl Acad Sci U S A
PNAS
Proceedings of the National Academy of Sciences of the United States of America
0027-8424
1091-6490
National Academy of Sciences

38498704
202315531
10.1073/pnas.2315531121
datasetDatasetresearch-articleResearch ArticleevolutionEvolution418
Biological Sciences
Evolution
Cip1, a CDK regulator, determines heterothallic mating or homothallic selfing in a protist
Ma Yang a 1 https://orcid.org/0000-0003-0772-6309

Yan Guanxiong a 1 https://orcid.org/0000-0001-6253-5922

Zhang Jing a https://orcid.org/0000-0002-7343-2184

Xiong Jie a b
Miao Wei miaowei@ihb.ac.cn
a c d e 2 https://orcid.org/0000-0003-3440-8322

aInstitute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China
bKey Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Chinese Academy of Sciences, Wuhan 430072, China
cCollege of Advanced Agricultural Sciences, University of Chinese Academy of Sciences, Beijing 100049, China
dKey laboratory of Lake and Watershed Science for Water Security, Chinese Academy of Sciences, Nanjing 210000, China
eInstitute of Hydrobiology, Hubei Hongshan Laboratory, Wuhan 430000, China
2To whom correspondence may be addressed. Email: miaowei@ihb.ac.cn.
Edited by Marcus Feldman, Stanford University, Stanford, CA; received September 8, 2023; accepted February 20, 2024

1Y.M. and G.Y. contributed equally to this work.

18 3 2024
26 3 2024
18 9 2024
121 13 e231553112108 9 2023
20 2 2024
Copyright © 2024 the Author(s). Published by PNAS.
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This article is distributed under Creative Commons Attribution-NonCommercial-NoDerivatives License 4.0 (CC BY-NC-ND).

Significance

Tracing the sexual reproduction status of the last eukaryotic common ancestor (LECA) and early eukaryotes poses a significant challenge because these species are now extinct and the available information on sexual reproduction in most extant eukaryotes is extremely limited. This study found that lack of the Cip1 protein in a free-living protist (belonging to Alveolate, which diverged from other major eukaryotic lineages over a billion years ago) leads to the initiation of sexual reproduction without the need for mating-type recognition. This finding provides insight into how selfing sexual reproduction might be controlled in the absence of mating types and supports the hypothesis that there might be an evolutionary stage in which ancient eukaryotes reproduced unisexually.

Mating type (sex) plays a crucial role in regulating sexual reproduction in most extant eukaryotes. One of the functions of mating types is ensuring self-incompatibility to some extent, thereby promoting genetic diversity. However, heterothallic mating is not always the best mating strategy. For example, in low-density populations or specific environments, such as parasitic ones, species may need to increase the ratio of potential mating partners. Consequently, many species allow homothallic selfing (i.e., self-fertility or intraclonal mating). Throughout the extensive evolutionary history of species, changes in environmental conditions have influenced mating strategies back and forth. However, the mechanisms through which mating-type recognition regulates sexual reproduction and the dynamics of mating strategy throughout evolution remain poorly understood. In this study, we show that the Cip1 protein is responsible for coupling sexual reproduction initiation to mating-type recognition in the protozoal eukaryote Tetrahymena thermophila. Deletion of the Cip1 protein leads to the loss of the selfing-avoidance function of mating-type recognition, resulting in selfing without mating-type recognition. Further experiments revealed that Cip1 is a regulatory subunit of the Cdk19–Cyc9 complex, which controls the initiation of sexual reproduction. These results reveal a mechanism that regulates the choice between mating and selfing. This mechanism also contributes to the debate about the ancestral state of sexual reproduction.

self-incompatibility
sexual reproduction initiation
mating-type recognition
ancestral eukaryotic sex
Tetrahymena
CAS | Bureau of Frontier Sciences and Education, Chinese Academy of Sciences (BFSE) 501100017670 ZDBS-LY-SM026 Wei Miao MOST | National Natural Science Foundation of China (NSFC) 501100001809 32130011 Guanxiong YanWei Miao MOST | National Natural Science Foundation of China (NSFC) 501100001809 32200344 Guanxiong YanWei Miao China Postdoctoral Science Foundation (China Postdoctoral Foundation Project) 501100002858 2021M703433 Guanxiong Yan
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pmcSexual reproduction is a prevalent mode of reproduction among eukaryotes and offers numerous advantages over asexual reproduction, such as increased genetic diversity and the opportunity for DNA damage repair (1–3). It plays an essential role in the life cycle of most species, even coexisting with asexual reproduction in some species. Most species employ a mating-type system (sex) to regulate sexual reproduction, ensuring self-incompatibility and thereby promoting genetic diversity. However, this system can also limit potential mates by half (in bisexual species), which can raise the potential of reproductive pressures, particularly in low-density populations or some specific environments (such as parasitic). Species in some lineages, such as some ciliates and basidiomycetes (4, 5), are multisexual, which reduces the pressure on mate availability. Also, some species allow selfing (i.e., self-fertility or intraclonal mating), enabling efficient sexual reproduction. Throughout the extensive evolutionary history of eukaryotes, changes in environmental conditions have influenced mating preferences and strategies. The cooperation between sexual selection and natural selection may drive rapid speciation. However, the mechanisms through which mating-type recognition regulates sexual reproduction and the dynamics of mating strategy throughout evolution remain poorly understood.

The origin of eukaryotic sexual reproduction is open to debate. The widespread existence of sexual reproduction in eukaryotes suggests that the last eukaryotic common ancestor (LECA) reproduced through meiotic sex. However, determining the origins and evolution of ancient sexual reproduction presents a significant challenge. Recent studies across diverse lineages reveal variations in sexual reproduction modes, shedding light on this fundamental question (reviewed in refs. 6–10). Complete eukaryotic meiotic sex involves events including ploidy alternation through meiosis, cell–cell fusion, and self/nonself recognition through mating types. Molecular foundations for meiosis (Spo11, Mre11, and so on) and cell–cell fusion (Hap2) appear relatively conserved across eukaryotes, suggesting an early origin. These events ensured ploidy alternation in most extant species. However, during the evolution of early eukaryotes, it remains unknown which event originated first. Both events may have alternative methods, with endoreduplication increasing ploidy and chromosome loss decreasing ploidy. Before the origin of meiosis and cell–cell fusion, early eukaryotes may alter their ploidy by these two methods, i.e., parasexual (SI Appendix, Fig. S1). Notably, parasexual reproduction has been reported in fungi like Aspergillus niger and Candida albicans (11, 12), indicating the feasibility of this mode.

In comparison to meiosis and cell–cell fusion, mating-type systems exhibit significant diversity among extant species, indicating independent origins in different lineages or a common origin followed by evolutionary diversification. Prior to the emergence of mating types, it is possible that species underwent a unisexual stage in which they obtained the capacity for sexual reproduction without exhibiting self-incompatibility (i.e., they could self) (SI Appendix, Fig. S1). Previous reports indicate that many species of eukaryotic microbial pathogens from various lineages [such as Cryptococcus neoformans (13) and Trypanosoma brucei (14)] have the ability of selfing, even though it appears to be a derived trait adapted to a parasitic lifestyle. It is worth noting that three mechanisms of extant selfing behavior can be proposed: i) the expression of two or more mating-type specificities in each individual (15, 16) or within the clone population (17); ii) a disturbance in mating-type recognition, leading to transmission of the wrong mating signal (18); or iii) the uncoupling of mating-type recognition from the initiation of sexual reproduction. The first two mechanisms still depend on mating-type recognition, whereas the third is independent. Therefore, only selfing behavior based on the third mechanism can be considered to provide evidence for ancestral unisexual species. However, due to a limited understanding of the molecular mechanisms underlying mating-type recognition and sexual reproduction initiation in most species (particularly free-living unicellular species), the experimental evidence is lacking to support or refute the existence of ancient unisexual species. It may provide insight into this debate to explore how selfing sexual reproduction could be regulated in the absence of mating types.

Tetrahymena thermophila (hereinafter referred to as Tetrahymena) is a free-living ciliate that belongs to Alveolate, which diverged from other major eukaryotic lineages over a billion years ago (19–21), and the Tetrahymena genus originated more than 300 Mya (22). Many well-known parasites, including Ichthyophthirius, Plasmodium, Toxoplasma, and Cryptosporidium, belong to Alveolate, and little is known about their mating recognition signal pathway. Tetrahymena has seven mating types (I to VII). Under conditions of adequate nutrition, cells reproduce asexually; after starvation, cells of any two different mating types can form pairs (conjugation), but those of the same mating type are unable to mate (Fig. 1A and SI Appendix, Fig. S2A) (23–25). We previously discovered that in Tetrahymena, the mating type is determined by a pair of genes, MTA and MTB (SI Appendix, Fig. S2B) (26). Both genes encode integral membrane proteins with extracellular mating-type-specific regions that mediate self and nonself mating-type recognition. Recently, we identified a sexual cyclin-dependent kinase (CDK) protein complex containing Cdk19 (a CDK) and Cyc9 (a cyclin) that is an essential regulator for the initiation of sexual reproduction in Tetrahymena (27). The expression patterns of CDK19 and CYC9 correlate highly with those of MTA and MTB (SI Appendix, Fig. S2C), and the proteins localize to the oral area (including the conjugation junction). Deletion of either CDK19 or CYC9 abolishes mating.

Fig. 1. CIP1-deleted cells undergo selfing after starvation. (A) After starvation, cip1Δ cells form pairs, despite having only one mating type (red dashed box). To distinguish between cell types, WT cells of mating type II (WT-II) were stained with MitoTracker Red. The same phenotype was observed in two independent cip1Δ cell lines of mating type VI and two of mating type VII (SI Appendix, Fig. S3B). (B) Pairing ratios for starved WT cells (black line), cip1Δ cells (red line), and a mixture of WT cells of two mating types (gray dashed line).

In this study, we found a key regulator, Cip1, that couples mating-type recognition to sexual reproduction initiation to prevent selfing. Sexual reproduction of CIP1 deletion (cip1Δ) strain is no longer controlled by mating-type recognition but directly initiated by the downstream Cdk19-Cyc9 complex. This finding sheds lights on the questions such as origins of self-incompatibility and sexual reproduction.

Results

Lack of Cip1 Transforms Tetrahymena From a Multisexual to a Unisexual Species.

We previously identified a protein that interacts with both Cdk19 and Cyc9, which we named Cip1 (CDK-cyclin-interacting protein, TTHERM_00891190) (27). Protein domain analysis predicted that Cip1 contains C-terminal WD40 repeats (SI Appendix, Fig. S2D). The CIP1 gene is strongly coexpressed with CDK19, CYC9, and the mating-type genes (MTA and MTB) (SI Appendix, Fig. S2C). To determine whether Cip1 regulates the initiation of sexual reproduction (similar to Cdk19 and Cyc9), CIP1 deletion strains were constructed. The morphology (Fig. 2A) and doubling time (SI Appendix, Fig. S3A) of cip1Δ cells were similar to those of wild-type (WT) cells under conditions of adequate nutrition. However, intraclonal mating (hereafter referred to as selfing) occurred after starvation (Fig. 1A, red dashed box). Selfing pairs could be observed at 4 h after starvation and the pairing rate reached ~70% within the next hour (Fig. 1B, red line). This is similar to the pairing rate between cells of different mating types (hereafter referred to as mating; Fig. 1B, dashed gray line).

Fig. 2. Cip1 negatively regulates sexual reproduction initiation. (A) Freshly starved WT-VII, WT-VI × WT-VII, and cip1Δ cells show no obvious morphological differences. (B–D) WT-VI × WT-VII and cip1Δ cells display the hallmarks of costimulation: tip transformation (B), cell pairing (C), and meiosis initiation (D). These events do not occur in WT-VII cells. Red dashed circles, cell tips; yellow arrows, elongated MIC during meiosis. Nuclei were visualized by DAPI staining. (E) Genes involved in mating, membrane fusion, and meiosis have similar expression patterns in WT-VI × WT-VII and cip1Δ cells. S4 (S5), starvation for 4 h (5 h); C1 (C2), conjugation for 1 h (2 h). (F) Schematic diagram showing MTT1-CIP1 construction. CIP1 expression is under the control of the Cd2+-inducible MTT1 promoter. BSR, blasticidin resistance selection cassette. (G) Selfing and mating ratios at different Cd2+ concentrations for equal numbers of MTT1-CIP1 and WT cells of different mating types. Pairing ratios were measured at 6 h after starvation.

Early Sexual Reproduction Events Are Normal in cip1Δ Selfing.

We next investigated whether early mating events occur normally in cip1Δ selfing pairs. The mating process in Tetrahymena is illustrated in SI Appendix, Fig. S2A. When WT cells mate, they first undergo a preconjugation stage (called costimulation), which involves a series of changes (28–31), such as tip transformation (Fig. 2B, 2, dashed red circle) and appearance of the concanavalin A receptor (ConA-R; SI Appendix, Fig. S4A, arrow). The cells then form loose pairs (Fig. 2C, 2), which is followed by tight pairing (SI Appendix, Fig. S4B) and the initiation of meiosis, during which the germline micronuclei start to elongate (Fig. 2D, 2, arrows).

We found evidence that suggests the selfing in cip1Δ cells is a canonical sexual reproduction process that resembles that occurring during mating: 1) Progress normally through the costimulation phase, as indicated by tip transformation (Figs. 2B and 3, dashed red circle) and ConA-R appearance (SI Appendix Fig. S4A, arrow). Mating-related genes were also expressed similarly in WT and cip1Δ cells during this stage (Fig. 2E). 2) Form loose pairs that gradually tightened, although this process was slower in cip1Δ cells (SI Appendix Fig. S4B). The temporal expression pattern of HAP2, a key regulator of membrane fusion during this process (32, 33), was similar in both WT and cip1Δ cells (Fig. 2E). 3) Initiate meiosis, as indicated by DAPI staining showed elongation of the germline nuclei (Figs. 2D and 3, arrows), and RNA-Seq analysis showed the expression of meiosis-related genes (Fig. 2E) (27, 34). In summary, these findings indicate that the early events of sexual reproduction occur normally in most cip1Δ selfing pairs.

Fig. 3. cip1Δ selfing is independent of mating-type recognition but requires the Cdk19–Cyc9 complex. (A) RNA-Seq reads mapped only to the mating-type-specific region of mating-type VII gene. MT, mating type. (B) Pairing ratio for WT cells, mutant cells, or a cell mixture at 5 h poststarvation. *, only mating pairs were counted. (C) Physical interaction among Cip1, Cdk19, and Cyc9 proteins. Numbers beside the arrows indicate the number of matched peptide spectra for each experiment. Data for Cdk19 and Cyc9 under starvation, costimulation, and conjugation conditions were taken from our previous study (27). (D) Western blot analysis of Cdk19-HA cross-link-IP. M, marker. Arrowhead, band of the expected size for CDK19-HA and associated complexes. *, band identity confirmed by MS (Dataset S2). (E) Identification of orthologs of mating-related genes. MTA and MTB orthologs are present in all but the most distantly related Tetrahymena species. CIP1 orthologs are present in all Tetrahymena species. CDK19 and CYC9 orthologs are present in all Tetrahymena species, and also in Paramecium. Orange dots, orthologous gene that has been reported (22) in the corresponding species; Gray dots, genes are similar but have low identity (sequence identity is shown below the dot). T. th, Tetrahymena thermophila; T. bo, Tetrahymena borealis; T. pi, Tetrahymena pigmentosa; T. pa, Tetrahymena paravorax; P. te, Paramecium tetraurelia.

We also observed that ~10% of cip1Δ cells formed pairs with abnormal morphology, with pairing occurring in dividing cells (SI Appendix, Fig. S4C). This suggests that another Cip1 function may be to arrest the cell cycle at the correct stage before the initiation of sexual reproduction. In addition, we observed arrested conjugation at the nucleus development stage in cip1Δ selfing pairs (SI Appendix, Fig. S4D), indicating a potential role for Cip1 in late conjugation events, such as internal eliminated sequences (IES) elimination, since defective IES elimination leads to arrest at this stage.

To investigate whether cip1Δ cells retain the ability to mate with WT cells of another mating type, we mixed equal numbers of cip1Δ and WT cells of different mating types and simultaneously starved them. Under these conditions, most cip1Δ cells formed selfing pairs (~81%), whereas a minority formed mating pairs with WT cells (~19%; SI Appendix, Fig. S4E). The observed selfing ratio was significantly higher than the expected ratio of ~33% under a no-selfing-mating preference. Our findings indicate that although cip1Δ cells can still mate with WT cells, they are more likely to undergo selfing.

CIP1 Regulates the Decision between Mating and Selfing.

To examine the correlation between mating/selfing ability and CIP1 expression level, an MTT1-CIP1 strain was constructed by replacing the CIP1 promoter with the Cd2+-inducible MTT1 promoter (Fig. 2F). MTT1-CIP1 and WT cells of different mating types were mixed at a 1:1 ratio and starved in the presence of Cd2+ at a range of concentrations. The total pairing ratios (selfing + mating) were similar at Cd2+ concentrations of 0 to 10 ng/mL. However, within this range, a higher proportion of MTT1-CIP1 cells underwent selfing at lower Cd2+ concentrations and more MTT1-CIP1 cells underwent mating at higher Cd2+ concentrations. When MTT1-CIP1 cells were exposed to 100 ng/mL Cd2+, the pairing ratio was significantly reduced and no selfing pairs were observed (Fig. 2G). These findings confirm that Cip1 is an important factor that regulates the decision between mating and selfing.

Selfing Behavior Is Independent of Mating-Type Recognition.

We next investigated whether the cip1Δ selfing strain resembles the hypothesized ancestral unisexual species. In the unisexual stage (prior to the origin of mating type), species directly initiated sexual reproduction without the need for mating-type recognition. However, as introduced before, there are also another two possibilities that can result in selfing but still rely on mating-type system.

To test these possibilities, we first investigated which mating-type genes are expressed in the cip1Δ strain (derived from a mating-type VII strain). RNA-Seq showed that no reads mapped to mating-type-specific regions II–VI, and relevant reads mapped only to region VII (Fig. 3A, dashed frame). Therefore, the selfing phenotype of cip1Δ does not result from the expression of mating-type genes of two or more specificities.

Next, we constructed double-deletion strains for CIP1 and each of the two mating-type genes (mtaΔcip1Δ and mtbΔcip1Δ). MTA and MTB genes are essential for mating-type recognition, and strains that lack either gene fail to mate (26). Both double-deletion strains showed a clear selfing phenotype but were unable to mate with WT cells (Figs. 2 and 3B). These results demonstrate that disabling mating-type recognition does not affect the selfing phenotype. Therefore, the selfing behavior is independent of mating-type recognition and resembles the ancestral unisexual stage.

Cip1 Regulates Sexual Reproduction Initiation through Cdk19-Cyc9.

We explored the mechanism by which Cip1 regulates sexual reproduction initiation based on our previous findings that a physical interaction between Cip1 and the Cdk19–Cyc9 complex, which is essential for mating (27). Thus, we hypothesized that Cip1 may regulate the initiation of sexual reproduction through the Cdk19–Cyc9 complex.

Reciprocal immunoprecipitation-coupled mass spectroscopy (IP-MS) analysis confirmed that Cip1 interacts with Cdk19 and Cyc9 at all three stages: starvation, costimulation, and conjugation (Fig. 3C and Dataset S1). To determine whether these proteins exist in a single complex, we cross-linked Cdk19-HA [hemagglutinin (HA) labeled] cells before IP and then analyzed the products by Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting. This showed the presence of three bands in addition to the ~36 kDa band corresponding to the molecular weight of Cdk19 (Fig. 3D): one of ~90 kDa, corresponding to Cdk19 + Cyc9; one of ~120 kDa, corresponding to Cdk19 + Cip1; and one of ~174 kDa, corresponding to Cdk19 + Cyc9 + Cip1 (also confirmed by MS; Dataset S2). These results suggest that Cip1 is a constitutive subunit of the Cdk19–Cyc9 complex. In addition, IP-MS analysis showed that the interaction between Cdk19 and Cyc9 persists when the CIP1 gene is deleted (Fig. 3C), revealing that assembly of the Cdk19–Cyc9 complex is not Cip1 dependent.

We then constructed double-deletion strains of CIP1 together with CDK19 or CYC9 (cdk19Δcip1Δ and cyc9Δcip1Δ) and performed mating tests. The results showed that both strains have the same phenotype as strains with a single CDK19 or CYC9 deletion, that is, they cannot undergo either selfing or mating (Fig. 3B, 3). We propose that Cip1 blocks the initiation of sexual reproduction via acting as a regulatory subunit of the Cdk19–Cyc9 complex.

In starved cells, immunofluorescence analysis showed a weak Cip1 signal, with undetectable Cdk19 and Cyc9 (SI Appendix, Fig. S5). In costimulated cells, all three proteins were detected in the cytoplasm (with Cdk19 also detected in the nucleus) and enriched at the cell tip (SI Appendix, Fig. S5, red arrows). In conjugating cells, all three proteins were enriched at the pairing junction (SI Appendix, Fig. S5, red arrows). Enrichment at the cell tip and pairing junction is consistent with a function for the Cdk19–Cyc9–Cip1 complex in mating regulation. In addition, Cip1 may be expressed before the other two proteins to prevent selfing via lack of Cip1 regulation of the Cdk19–Cyc9 complex. In addition, analysis of orthologous proteins in related species suggests that mating-type proteins evolved after Cdk19 and Cyc9 (Fig. 3E). Therefore, the Tetrahymena ancestor may have initiated sexual reproduction via the Cdk19–Cyc9 complex but without mating-type recognition through Mta and Mtb.

Discussion

Our results revealed a mating recognition signal pathway, demonstrating that Tetrahymena couples mating-type recognition to sexual reproduction initiation through Cip1, which regulates the Cdk19–Cyc9 complex and, thereby, prevents selfing (Fig. 4A). We showed that the mating strategy, whether mating or selfing, can be shifted by the expression level of CIP1. Additionally, this finding in ciliates, a eukaryotic lineage distant from animals, plants, and fungi, provides insight into the origin and evolution of mating-type/self-incompatibility system.

Fig. 4. Cip1 couples mating-type recognition to sexual reproduction initiation in Tetrahymena. (A) In starved WT cells, Cip1 regulation of the Cdk19–Cyc9 complex prevents sexual reproduction initiation. When cells of different mating types recognize each other, the Mta–Mtb interaction releases Cip1 inhibition, allowing sexual reproduction (mating) to proceed through Cdk19–Cyc9. In cip1Δ cells, mating-type recognition is uncoupled from sexual reproduction. Direct initiation of sexual reproduction (selfing) is independent of Mta and Mtb since Cdk19–Cyc9 is not inhibited. (B) CIP1 deletion abolishes mating-type recognition and transforms Tetrahymena into a unisexual species, thus reversing the origin of mating types.

Shifts in Mating Strategies across Evolution.

The mating strategy of selfing, inbreeding or outcrossing plays an important role in a species’ adaptation to varying environmental conditions. The Tetrahymena genus, believed to have originated over 300 Mya (22), predating the separation of Pangaea (~200 Mya), has experienced numerous environmental shifts. As previously discussed (16, 25), within this genus, there is evidence of mechanisms supporting inbreeding, such as multisexual systems and karyonidal mating-type determination in the Borealis clade. Conversely, outcrossing is also facilitated through a spectrum of adaptations, including a long sexual immaturity period, intranuclear coordination during mating-type determination, allelic assortment, and synclonal mating-type determination in the Australis clade. These dual strategies reflect a delicate equilibrium and are suggestive of a complex interplay driven by an ever-changing environment throughout the lineage's evolution.

In this study, we identified a mechanism capable of transforming Tetrahymena from a multisexual species to unisexual. To our knowledge, this form of selfing has not been found before. It is surprising that this transition is triggered by the deletion of a single gene. Within extant Tetrahymena, beyond its role in regulating mating-type recognition, this gene was identified as a critical player in other developmental events, such as cell cycle arrest and IES elimination. In addition, compared with other features which promote inbreeding, lacking CIP1 results in a preference of selfing, and this selfing is potentially irreversible. Owing to the challenges associated with their survival and adaptation, it is reasonable that such selfing strains may not exist in nature.

Alternation of mating strategy is common in other species. In unicellular species, such as Saccharomyces cerevisiae, the transition between homothallic (selfing) and heterothallic (outcrossing) modes is naturally orchestrated by the presence or absence of the HO mating-type switch system (17). Many parasitic protists and fungi, including C. neoformans and T. brucei (6, 10, 13, 14), exhibit the ability of selfing, which may serve as an adaptive response to the unique reproductive challenges posed by parasitic lifestyles. It has been reported that some multicellular plants alternate between self-mating and obligate outcrossing [such as Arabidopsis (35–37) and Solanum species (38)]. Transformation from unicellular to multicellular involves considerable changes to mating systems. One such change is the loss of efficacy of self-incompatibility which was established by a previous mating-type system, because individual may generate gametes of different mating types (hermaphrodite). To solve this problem, more mechanisms need to be employed to regulate self-incompatibility, such as becoming dioecious or evolving a novel self-/nonself recognition mechanism (SI systems). Selective pressures on sexual reproduction can drive alterations in protein function at the SI locus, resulting in alternation between selfing and outcrossing. In multicellular animals, mechanisms for selfing avoidance are more complicated. In addition to dioecious and SI systems, the regulations on behavioral level also play important roles.

It is noteworthy that cip1Δ can engage in both homothallic selfing and heterothallic mating. In addition to completely deletion, the MTT1-CIP1 results showed that regulating the expression level of CIP1 balance the decision between mating and selfing. In this context, interesting population genetics questions can be raised: Is this form of the mating system stable during evolution? In different environments, does this mating system evolve toward selfing or mating (how does natural selection interact with sexual selection)? It is plausible that a mating mode like cip1Δ is an intermediate evolutionary stage, with subsequent natural selection driving the mating strategy to evolve toward the most appropriate one. In fact, although the mechanism is different, this mating mode has been reported in C. albicans, an opportunistic human fungal pathogen (39).

Another question is whether this system promotes/influences speciation. In the absence of Cip1, the mating-type recognition system becomes redundant, reducing selection pressure on relevant molecules and making them easy to loss or change. Extreme examples are mtaΔcip1Δ and mtbΔcip1Δ. These strains cannot mate with WT Tetrahymena but still can initiate selfing, i.e., it established reproductive isolation with WT Tetrahymena and could be an origin of a new species. This suggests a potential mechanism of how changes in mating strategy (sexual selection) contribute to rapid speciation. Whether this hypothesized process has occurred in nature remains unknown. Further simulations and experimental population genetics studies in various lineages may examine this possibility and contribute to a general theory.

Selfing in cip1Δ Tetrahymena Cells Supports the Unisexual LECA Hypothesis.

Two important innovations were necessary to enable complete eukaryotic meiotic sex: i) ploidy alternation via cell–cell fusion and meiosis and ii) a self/nonself recognition mechanism coupled to sexual reproduction initiation, that is, the origin of mating types to prevent selfing (i.e., self-fertility or intraclonal mating) (6, 8, 9). While both the ploidy alternation cycle via meiosis and cell–cell fusion and mating-type systems are prevalent in most extant eukaryotes, their molecular bases exhibit striking differences in terms of conservation. In the case of meiosis and cell–cell fusion, both the process and associated proteins show remarkable conservation. For example, DSB formation (by Spo11), DSB repair (involving Com1, Dmc1, Hop2 etc.), chromosomal cross-over (involving Msh4, Msh5, Sgs1 etc.), chromosome condensation and cohesion (involving Smc2, Smc4, Smc1, Smc3 etc.). However, a striking variety of mating-type system (signaling molecules, downstream pathways, and expression regulators) are found in different supergroups: Take signaling molecular for example, in Fungi, mating-type recognition involves GPCR and their pheromone ligands. While in Ciliate, the mating-type proteins of Tetrahymena are two membrane proteins containing GFR domain (Mta and Mtb) without the involvement of pheromones; hypotrich Ciliate Euplotes utilize two proteins derived from intron-splicing variants of the same gene that embody ligand and receptor function (40, 41); in heterotrich Ciliate Blepharisma, the mating-type ligand is a small tryptophan-related molecule (42, 43). Therefore, it is reasonable to infer that meiotic sex was already present in the LECA, while mating-type systems originated later in different lineages independently. LECA and early eukaryotes might have relied on meiotic sex to repair DNA damage caused by mitochondrial reactive oxygen species, but they may not have used this process to promote genetic diversity because selfing was not restricted. This leads to a hypothesis that the LECA and early eukaryotes were unisexual (SI Appendix, Fig. S1) (reviewed by refs. 6–9). It still cannot be excluded that a single mating-type system was present in the LECA and that this system evolved in subsequent radiations, during the process of speciation, into diverse forms. Conclusive evidence is still lacking to distinguish between these two possibilities.

The modern bisexual/multisexual state differs from the ancestral unisexual state in two important aspects: i) sexual reproduction takes place between any two individuals or only among those with different mating types, and ii) whether sexual reproduction is regulated by a mating-type recognition system. In Tetrahymena, it has been reported that mutant strains that express multiple mating-type genes (e.g., mating-type II and V genes) are capable of selfing (44). However, this selfing does not resemble the ancestral unisexual state because it still relies on the mating-type recognition system. Conversely, selfing in cip1Δ cells is independent of mating-type recognition, suggesting the existence of an ancient unisexual stage (Fig. 4B).

Few examples of free-living unicellular species that exhibit self-mating have been reported. Although some parasitic species undergo selfing, these forms of sexual reproduction are likely to be a consequence of evolutionary pressures linked to their particular life cycles. In contrast, our results demonstrate that cip1Δ Tetrahymena cells exhibit selfing behavior. The inherent features of Tetrahymena—its free-living nature, facultative sexual reproduction, and the presence of multiple mating types—mitigate the evolutionary pressures on sexual reproduction. Therefore, its potential for selfing in the absence of Cip1 may reflect reversion to an ancestral state. Our study presents evidence indicating that the ancestor of Tetrahymena, a genus that originated more than 300 Mya (22), is likely to be unisexual. However, the direct connection of this evolutionary unisexual stage to LECA remains uncertain. While our findings support the hypothesis that LECA could have been unisexual, they do not conclusively prove it.

CDK Inhibitor and Sexual Reproduction Regulation.

Protein domain prediction revealed that the distinct feature of Cip1 is a C-terminal region of ~330 amino acids containing WD40 repeats. Despite relatively low sequence similarity (~10%), this feature aligns with the canonical domain of Fizzy family proteins (SI Appendix, Fig. S6). Such proteins typically inhibit the CDK–cyclin complex and, consequently, induce cell cycle arrest (including sexual cell cycle arrest) (45–48). A well-studied example is the Schizosaccharomyces Srw1 protein, which negatively regulates the Cdc2–Cdc13 complex to induce G1 arrest before mating in response to nitrogen starvation (45). Comparable mechanisms have been observed in Mus species, in which CDH1 negatively modulates CDK1–cyclin B1, leading to G2 arrest of meiosis I in oocytes (46). Our findings suggest that Cip1 is a regulatory subunit of Cdk19–Cyc9, resulting in the arrest of sexual reproduction initiation. Moreover, Fizzy family proteins are essential for sexual reproduction in Plasmodium and Arabidopsis species (47, 48).

The widespread utilization of CDK and cyclin proteins to regulate the eukaryotic cell cycle suggests that this regulatory mechanism originated in the LECA. To ensure precise cell cycle regulation, CDK activity is tightly controlled by regulators in addition to cyclin, including the CDK-activating kinase, CDK regulatory subunit, and CDK inhibitory subunit. Although the exact method for regulation of sexual cell cycles in the LECA and early eukaryotes before the emergence of mating types remains unknown, the involvement of CDK and its regulators is plausible. Our findings offer insight into how regulation of the CDK complex may control selfing in the absence of mating types.

The abundance of WD40 proteins across diverse organisms presents a challenge to fully comprehending their functional and evolutionary connections, particularly considering the vast evolutionary timeline and diverse ways in which eukaryotes initiate sexual reproduction and mating-type recognition. Future genomic and functional studies across a broad taxonomic spectrum may reveal whether similar mechanisms exist in other eukaryotes and provide more evidence to support a unisexual state for the LECA.

Materials and Methods

Strains and Culture Conditions.

T. thermophila WT strains CU427 (mating type VI), CU428 (mating type VII), and B2086 (mating type II) were obtained from the Tetrahymena Stock Center (http://tetrahymena.vet.cornell.edu/). Cells were cultured in Super Proteose Peptone (SPP) medium (1% proteose peptone, 0.2% glucose, 0.1% yeast extract, and 0.003% Sequestrene) (49). When cells reached logarithmic phase (at ~2 × 105 cells/mL), they were starved by washing once in 10 mM Tris-HCl (pH 7.4) and culturing in 10 mM Tris-HCl.

Somatic Gene Deletion and Protein Tagging.

To construct CIP1 deletion strains, one DNA fragment (~1 kb) upstream of the open reading frame and one downstream fragment (~1 kb) were amplified using the primers listed in Dataset S3. The two fragments were ligated to a CHX cassette, containing a cycloheximide resistance gene driven by a Cd2+-inducible MTT1 metallothionein promoter. The deletion construct was obtained by restriction endonuclease digestion and introduced into starved WT CU427 and CU428 cells by biolistic transformation (50). Transformants were selected in SPP medium containing CdCl2 and cycloheximide until all ~90 WT copies were replaced by deletion copies (51). Gene deletion was confirmed by PCR. To acquire double-deletion strains, the CIP1 deletion plasmid was transformed into the mtaΔ, mtbΔ, cdk19Δ, and cyc9Δ strains.

C-terminally HA-tagged Cip1 strains were constructed using an insertion method: one fragment (~500 bp) downstream of CIP1 and one fragment on the CIP1-coding region (~1 kb) were amplified and recombined through the HA-homologous arm. The recombined fragment and one fragment (~1 kb) downstream of the earlier-mentioned 500 bp fragment were ligated to pNEO4. Transformants were selected using decreasing CdCl2 and increasing paromomycin concentrations.

MTT1-CIP1 Construction.

One DNA fragment (~1 kb; Fig. 2F, frag1) upstream of the open reading frame and one fragment of the first 1.3 kb of the CIP1 genomic sequence (Fig. 2F, frag2) were amplified. The Cd2+-inducible MTT1 promoter was amplified from pNEO4 and fused first to fragment 2 and then to fragment 1 and a blasticidin-resistance cassette (BSR) from pMTT1-GFP-BSR. Finally, the entire fragment was cloned into the pBlueScript SK (+) backbone.

Mating Experiments.

Equal proportions of cells of two different mating types were starved for 16 to 18 h and then mixed to obtain conjugating cells. For testing cip1Δ cells, the percentage pairing was calculated at the time after starvation. The two types of cells were distinguished by overnight starvation (or culture in SPP medium) in the presence of 500 nM MitoTracker Red-CMXRos (Invitrogen, Eugene, OR, USA). Cells were then washed twice in Tris-HCl (pH 7.4) and resuspended at approximately 2.5 × 105 cells/mL. A total of 300 cells or pairs were counted (in triplicate, error bar: SD). The percentage pairing was calculated as:Percentage pairing (%) = (pairs × 2)/[single cell + (pairs × 2)] × 100.

Detection of Costimulation Events.

Fluorescein ConA labeling was performed as previously described (27, 31). Briefly, samples were prepared and fixed in 4% paraformaldehyde (PFA) in 0.1 M cold phosphate buffer for 5 min and then washed three times. Cells were then incubated with 10 μg/mL fluorescein-labeled ConA (Vector Laboratories, Burlingame, CA, USA) for 5 min on ice, washed three times, and then observed by fluorescence microscopy (Olympus BX51). Images were taken at 40× magnification.

Tip transformation was tested at the starvation and costimulation stages (after mixing cells for 1 h). Samples were observed immediately at 20× magnification by light microscopy and photographed (Olympus BX51).

Tight Pairs Test.

Starved cells of two mating types were mixed at a 1:1 ratio and cultured at 30 °C. Samples were taken at 15-min intervals; for this, 200 μL of each sample was placed in a 1.5 mL centrifuge tube. From each sample, 20 μL cells were stained with Lugol’s iodine, and the remainder were vortexed for 15 s (Vortex-5, Qilimbel, China) and then fixed in Lugol’s iodine. The cell pairing rate before and after vortexing was calculated.

Immunofluorescence Analysis.

For this analysis, 5 mL cells (∼2 × 105 cells) from the starvation, costimulation, and conjugation stages were prepared as previously described (52). An 80 μL sample of the cell suspension was spread onto a slide and air-dried in a fume hood. Slides were then washed twice with 1× phosphate-buffered saline (PBS) and once with phosphate-buffered saline with Triton X-100 (PBST, PBS containing 0.05% Triton X-100), and then incubated in blocking buffer (5% bovine serum albumin (BSA) and 2.25% glycine in PBST) for 30 min at room temperature to block nonspecific binding sites. The slides were then washed three times (twice with PBS and once with PBST) and incubated with mouse anti-HA antibody (1:100 dilution; clone 16B12, Covance, Berkeley, CA, USA) for 1 h. Next, slides were washed as described above, incubated in fluorescein isothiocyanate-labeled goat anti-mouse immunoglobulin (H+L, 1:1,000 dilution, SA00003-1, Proteintech Group, Chicago, IL) for 2 h at room temperature, and then washed three times. The slides were then mounted with anti-fading agent (Vector Laboratories) containing 0.5 mg/mL DAPI. Confocal images were acquired by laser scanning confocal microscopy (Leica TCS SP8, Leica Microsystems, Mannheim, Germany) and visualized by Leica LAS X software (https://www.leica-microsystems.com/products/microscope-software/p/leica-las-x-ls/).

IP-MS.

HA-labeled and WT cell samples at the starvation, costimulation, and conjugation stages were prepared and lysed in lysis buffer (30 mM Tris-HCl, 20 mM KCl, 2 mM MgCl2, 1 mM phenylmethylsulfonyl fluoride, and 0.1% Triton X-100) containing cOmplete proteinase inhibitor (Roche Diagnostics, Indianapolis, IN, USA) with ultrasound treatment. (For Cdk19-HA cross-link-IP, cells were first incubated with 0.6% PFA and then lysed). After centrifugation, the cell supernatant was incubated with anti-HA agarose beads (Sigma-Aldrich, St Louis, MO, USA) at 4 °C for 2 h. After washing with lysis buffer containing 150 mM NaCl, the HA peptide was added to elute target proteins from the beads. Liquid Chromatography-Tandem Mass Spectrometry analysis was performed to detect interacting proteins. High-resolution mass spectra of the peptide mixture were deconvoluted using the Xtract software (Thermo Scientific). Raw MS data were analyzed using Proteome Discoverer 2.1 software. To detect proteins in specific bands from cross-link-IP products, gel pieces of the corresponding size were cut and incubated with 50 mM DTT (10 min, at room temperature) and 50 mM NH4HCO3 (30 min, at room temperature) before MS.

Transcriptome Analysis.

Total RNA was extracted from WT and cip1Δ cells after starvation for 4 h and 5 h, respectively, using the RNeasy Protect Cell Mini Kit (Qiagen, Valencia, CA, USA). The RNA was sequenced and mapped as described previously (34). Sequencing data for conjugating WT cells (WT-C1 and WT-C2) were derived from NCBI (GSE80977).

Supplementary Material

Appendix 01 (PDF)

Dataset S01 (XLSX)

Dataset S02 (XLSX)

Dataset S03 (XLSX)

We thank Min Wang and Shuzhao Jia at the Analysis and Testing Center of Institute of Hydrobiology, Chinese Academy of Sciences for their help with mass spectrometry. The bioinformatics analysis was supported by the Wuhan Branch, Supercomputing Center, Chinese Academy of Sciences, China. We also want to thank members of the Protist 10,000 Genomes Project consortium for helpful suggestions. The culture and maintenance of Tetrahymena cells were supported by the National Aquatic Biological Resource Center. This study was supported by the Bureau of Frontier Sciences and Education, Chinese Academy of Sciences [ZDBS-LY-SM026] (to W.M.), the National Natural Science Foundation of China [32130011] (to W.M.) and [32200344] (to G.Y.), and the China Postdoctoral Science Foundation [2021M703433] (to G.Y.).

Author contributions

Y.M., G.Y., and W.M. designed research; Y.M., G.Y., and J.Z. performed research; Y.M., G.Y., and J.X. contributed new reagents/analytic tools; Y.M. and G.Y. analyzed data; J.X. helped to conceptualize the work; W.M. supervised the work; and Y.M. and G.Y. wrote the paper.

Competing interests

The authors declare no competing interest.

Data, Materials, and Software Availability

All study data are included in the article and/or supporting information.

Supporting Information

This article is a PNAS Direct Submission.
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