Life cycle strategies in free‐living unicellular eukaryotes: Diversity, evolution, and current molecular tools to unravel the private life of microorganisms

Variations in the process of syngamy: mating patterns, gamete morphologies, and mating types

The definition of sex is tightly associated with two cellular processes: meiosis and syngamy (Goodenough & Heitman, 2014). The most common pattern of syngamy implies the fusion of both the cytoplasm and the nuclei of two specialized sexual cells called gametes. This modality of syngamy has been documented in algal lineages such as Dinoflagellata, Ochrophyta, Chlorophyta, and Haptophyta (Figueroa & Rengefors, 2006; Lee et al., 2021; Nishimura, 2010; Rousseau et al., 2007). However, divergent syngamy patterns have also been reported (Bilcke et al., 2021; Ma et al., 2020; Preobrazhenskaya & Tarasova, 2004). For instance, gamete formation and syngamy can both occur inside vegetative cells, which pair to optimize gamete encounters. This mating pattern has been observed among Cercozoa (Arnold, 1966) and some Ochrophyta such as pennate diatoms (Bell, 1988; Bilcke et al., 2021), while some Foraminifera can form pairs of cells or even aggregates of up to four cells during mating (Preobrazhenskaya & Tarasova, 2004; Röttger, 1974). Alternatively, syngamy in Ciliophora occurs without the formation of gametes, but rather through the exchange and fusion of the nuclei of two vegetative cells, which subsequently undergo cytokinesis (Loidl, 2021; Ma et al., 2020).

Gametes can also be formed by life stages that differ morphologically from the vegetative stage. Two life stages that are frequently associated with sexual cycles among multiple protist lineages are cysts and colonial stages (Figure 1A). Cysts are widespread and probably evolved for allowing the cell to endure limiting or stressful environmental conditions (Figueroa & Rengefors, 2006). However, cyst formation during sex has been reported in some Amoebozoa (e.g. order Arcellinida, Lee et al., 2000) and TSAR lineages (i.e. the diverse clade including Telonemia, Stramenopiles, Alveolata, and Rhizaria, Figure 2) (Decelle et al., 2013; Okamoto et al., 2016). Moreover, cyst formation after gamete fusion has been described for Ochrophyta (Horiguchi, 2017), while gamete emergence from cysts has been documented among Dinoflagellata (Montagnes et al., 2011) and Endomyxa (the occurrence of meiotic stages in resting cysts has been suggested in 1993 for the species Lateromyxa gallica (Hess & Suthaus, 2022)) and is suspected in Acantharia (Radiolaria, Decelle et al., 2013). Cases where the colonial cycle is connected to the sexual cycle have been reported for Chlorophyta (Umen, 2020), Haptophyta have been proposed for Radiolaria (Yuasa & Takahashi, 2014) and are frequent among Amoebozoa (Dhakshinamoorthy & Singh, 2021; Flowers et al., 2010; Kin & Schaap, 2021). Nevertheless, there is a notable difference in the connexion between colonial and sexual cycles among these lineages. Amoebozoa and colonial Chlorophyta exit the colonial cycle by the vegetative life stage (Kin & Schaap, 2021; Umen, 2020), while colonial Haptophyta by the formation of gametes. Therefore, for haploid protists syngamy occurs at the beginning of the colonial cycle, while for diploid protists syngamy occurs at the end of the colonial cycle.

Compatibility between gametes is necessary for syngamy to occur. In some lineages gametes originating from the same vegetative cell can be compatible and undergo syngamy (i.e. autogamy or self‐fertilization), while in other lineages syngamy occurs only between gametes produced by different vegetative cells (i.e. heterogamy). Across protist evolution, autogamy has been documented among Amoebozoa (e.g. Eumycetozoa, Flowers et al., 2010) and has been conserved among a few representatives of some other lineages (e.g. Foraminifera (Goetz et al., 2022) and Ochrophyta (Ferrante et al., 2019)). However, autogamy remains scarce likely because it limits genetic diversification of the next generation and adaptation to changing conditions. On the contrary, heterogamy appears more widespread and is associated with many cellular and molecular innovations. In some heterogamous protists, different vegetative cells with the same morphology seem to fuse without the formation of gametes, a mating pattern that can be considered as ancestral (e.g. Amoebozoa (Flowers et al., 2010), Ciliophora (Loidl, 2021), Haptophyta, and Euglenozoa (sexuality observed for only one species, Lee et al., 2000)). This type of heterogamy between cells of the same morphology is referred to as isogamy and when the vegetative life stages are the ones that fuse, hologamy. In other protist lineages, isogamy occurs between gametes of the same morphology, usually being flagellated and smaller than the vegetative cells (e.g. Foraminifera, Cercozoa, Dinoflagellata, Chlorophyta, and Ochrophyta (Boltovskoy et al., 2017; Ferrante et al., 2019; Lee et al., 2021; Lehmann et al., 2006; Umen, 2020)). An elaborated trait of heterogamous fusion systems is the emergence of molecular discriminants among gametes originating from different or same vegetative cells (i.e. a concept commonly defined as heterothallism or homothallism among multicellular organisms (Beukeboom & Perrin, 2014)). These gamete molecular discriminants define different “mating types.” The number of mating types is highly variable across lineages, ranging from two among Chlorophyta (e.g. Chlamydomonas) (Nishimura, 2010), unicellular Fungi (e.g. Saccharomyces) (Hanson & Wolfe, 2017), and Ochrophyta (e.g. Skeletonema) (Ferrante et al., 2019), to three for social Amoebozoa (e.g. Dictyostellium) (Okamoto et al., 2016) and up to seven for Ciliophora (e.g. Tetrahymena) (Ma et al., 2020). In social Amoebozoa, hologamy between different mating types illustrates that syngamy traits that can be considered as ancestral (i.e. fusion without gamete formation) can co‐occur with elaborated heterogamous traits (i.e. mating type differentation). The most complex form of heterogamy is anisogamy, in which gametes of different mating types exhibit morphological discriminants, like size differences. Typically, a larger gamete carrying the resources for zygote development fuses with a smaller gamete. Anisogamy occurs among Ochrophyta (Montresor et al., 2016), Dinoflagellata (Montagnes et al., 2011), and Chlorophyta (Lee et al., 2000).

Variations in ploidy and the number of life phases

Ploidy variations in sexual life cycles are common among protists. The vegetative life stage of protists can either be diploid (i.e. life cycle characterized as diploid) or haploid (i.e. life cycle characterized as haploid) or both ploidies can alternate as different life phases in the life cycle (i.e. life cycle characterized as haploid‐diploid) (Beukeboom & Perrin, 2014; Frada, 2009; Frada et al., 2019) (Figure 1B). Sexual life cycles have been intriguing evolutionary biologists for decades (Coelho et al., 2007; Mable & Otto, 1998; Otto & Gerstein, 2008; reviewed in Valero et al., 1992). As nearly all metazoans develop somatically as diploids in the vegetative stage and in plants there is a reduction of the haploid phase with increasing developmental complexity, early studies pointed towards diploid superiority. Diploidy could confer genetic advantages of having two sets of homologous chromosomes, which can mask deleterious mutations (Crow & Kimura, 1965) while enhancing the fixation rate of beneficial mutations, thereby promoting adaptation (Paquin & Adams, 1983). However, the same arguments could be employed to explain the evolution of haploid life cycles (e.g. Crow & Kimura, 1965; Jenkins & Kirkpatrick, 1995; Otto & Goldstein, 1992; Otto & Marks, 1996). Deleterious mutations in haploid organisms would be purged out of populations rapidly, while new mutations could be expressed immediately and enable a quick generation of novel phenotypic variants, capable of adaptation to different environmental conditions. Haploid‐diploid life cycles were seen as a transitional state of likely low relevance (Mable & Otto, 1998). Nevertheless, within the free‐living protist lineages with described sexual cycles (15 lineages), ~30% of lineages include haploid life cycles (five out of 15), ~50% of lineages include diploid life cycles (eight out of 15), while ~50% of lineages (eight out of 15) include haploid‐diploid life cycles (Figure 2; Table S1, e.g. Amoebozoa, Haptophyta, Rhodophyta, Rhizaria, Alveolata). This indicates that the haploid‐diploid variants are still commonplace and prevalent in the modern eukaryotic lineages. It is thus plausible to envision that from an evolutionary viewpoint, haploid‐diploid life cycles were possibly the ancestral sexual life cycle strategy in early eukaryotes. The biphasic haploid‐diploid cycle would then have been modulated leading to the reduction of the prevalence of one of the ploidy phases (i.e. the haploid or the diploid), independently, in different lineages as organisms adapted to specific niches. In terms of sex, biphasic life cycles imply that meiosis and syngamy occur during different life phases. The diploid life phase that undergoes meiosis is usually called agamont, while the haploid life phase that undergoes syngamy is called gamont in reference to gametes (even though it has been cited above that gametes are not the only life stage that can undergo syngamy, e.g. hologamy (Ma et al., 2020)) (Figure 1B). The existence of a third life phase, called schizont, has been described for Foraminifera (Dettmering et al., 1998; Lehmann et al., 2006). Under some conditions the agamont produces a life phase that is morphologically similar to the haploid gamont, but that does not produce gametes. The schizont life phase can replicate itself asexually, while its ploidy and cellular processes mediating the transitions with the gamont and agamont life phases remain unclear (Darling et al., 2023; Dettmering et al., 1998; Lehmann et al., 2006). As the schizont life phase is occasional, it is thought to stand as a resting life phase triggered to potentially develop into a gamont according to the environmental conditions (Dettmering et al., 1998). The number of life cycle phases can vary between protists among the same lineage. For instance, all three types of life phases (i.e. diploid, haploid and haploid‐diploid) are encountered among Fungi (e.g. haploid Neurospora, diploid and haploid‐diploid Saccharomyces (Stajich et al., 2009)) and Dinoflagellata (e.g. haploid Lingulodinium, diploid Noctiluca, and haploid‐diploid Alexandrium (Figueroa et al., 2015; Figueroa & Bravo, 2005; Lee et al., 2021)) (Figure 2; Table S1). Generations can have the same or different morphologies, being either isomorphic (e.g. Choanoflagellata and Obazoa (Levin & King, 2013)) or heteromorphic (e.g. Myxogastria, Eumycetozoa (Everhart & Keller, 2008), Prymnesiophyceae, Haptophyta (Frada et al., 2019), and Foraminifera (Darling et al., 2023)).

Overall, sexual cycle strategies among protists likely evolved from haploid‐diploid life cycles with isogamous mating towards either haploid or diploid life cycles with anisogamous mating systems. Independent reduction of one of the life phases and various degrees of specialization during adaptation to specific environments probably resulted in the diversity of life cycles observed today (Figure 2). The combination of ancestral and elaborated life cycle traits could, thus, be linked to different life strategies adopted by each lineage according to the advantages they conferred in their ecological niche.

Variations in parameters triggering sexual cycles

The environment has a direct influence on the sexual cycle of protists. As asexual reproduction requires relatively less energy than binary fission, allocating energy and cellular resources for sex is worth the investment when it provides higher chances for survival than asexual division (Speijer, 2016). The most common sexual cycle triggers are stress signals coming from the environment (i.e. external triggers), such as a lack of nutrients. Nutrient depletion is a sexual cycle trigger across various protist lineages like social Amoebozoa (Dunn et al., 2018; Flowers et al., 2010), Ciliophora (Ma et al., 2020), Dinoflagellata (Lee et al., 2021; Montagnes et al., 2011), Ochrophyta (Figueroa & Rengefors, 2006; Nanjappa et al., 2017), and Chlorophyta (Huang & Beck, 2003). For photosynthetic protists, light exposure time can also be an inducing factor (e.g. Haptophyta (Rousseau et al., 2007), Ochrophyta (Mouget et al., 2009), and Chlorophyta (Huang & Beck, 2003)), while for marine protists salinity changes have also been shown to play a role in triggering their sexual cycle (e.g. Ochrophyta (Ferrante et al., 2019)). In cultivable lineages, the effect of multiple external triggers, such as temperature and simulated wave motion, can be tested (e.g. Dinoflagellata, Lee et al., 2021). However, sexual cycle triggers in uncultivable lineages remain hypothetical and general, often linked to seasonal patterns (e.g. reproduction in Radiolaria has been hypothesized to follow seasonal variations in prey availability, Decelle et al., 2013). Seasons and moon phases have been proposed as regulators of the sexual cycle among Foraminifera, both in culture and field observations (Hohenegger et al., 2019; Lončarić et al., 2005; Spindler et al., 1979). Triggers initiating the sexual cycle can also originate from the cell itself (i.e. internal factors). Diurnal gene expression patterns have led to the molecular description of internal clocks for protists among Chlorophyta (e.g. Ostreoccoccus, Monnier et al., 2010) and Stramenopiles (e.g. Ochrophyta, Farré, 2020). Similar to multicellular organisms, pheromone production is also encountered among protists. Sex pheromones (also called gamones) have been identified in gametes of social Amoebozoa (O'Day & Keszei, 2012), Haptista (Rousseau et al., 2007), Chlorophyta (Lee et al., 2000), and Ochrophyta (Bilcke et al., 2020; Ferrante et al., 2019). Cell population aspects like cell size and cell concentration have also been found to induce sex in some Ochrophyta and Dinoflagellata (Bilcke et al., 2020; Ferrante et al., 2019; Lee et al., 2021). Even though laboratory conditions provide precious insight into factors inducing life cycle transitions among protists, they might not reflect the lifespan and sex frequency of organisms in nature. Monitoring environmental protist populations provides valuable information that cannot be extrapolated from in vitro observations. For instance, it has been shown that benthic Foraminifera sexual cycle occurs all year round and can be up‐regulated by external factors (e.g. higher temperature and nutrient concentration) (Hohenegger et al., 2019).

A minority of data available: the best is yet to come

Overall, knowledge of sexual cycles among free‐living protists is fragmentary, has been confirmed only among cultivable species (Figure 2; Table S1) and indicates a latent potential for describing diverse sexual cycle protist strategies. In the eukaryotic tree of life by Richter et al. (2022), the disparity of data availability across eukaryotic lineages reflects the scarcity of knowledge regarding free‐living protist life cycles (Figure 2; Table S1). Among the lineages that include protist representatives (71 out of 72 lineages, only metazoa being exclusively multicellular), the protist lineages with free‐living representatives correspond to 66, out of which ~20% (15 lineages) have a confirmed sexual cycle (i.e. both meiosis and syngamy observed in vitro). In few cases, sexual cues have been partly observed with cytological observations (i.e. cellular processes involved in sexual cycles) and/or genetic patterns (i.e. indices in population genetics structure and presence of sex‐related genes) or have been suggested based on life cycle similarities with known sexual groups (e.g. radiolarian swarmers are morphologically similar to foraminiferan gametes (Decelle et al., 2015; Yuasa & Takahashi, 2014, 2016)). Yet, evidence of sexual processes is completely lacking for ~45% (30/66 lineages) of free‐living protist lineages, many of which are associated with few genetic data available (Figure 2). To date, no exclusively asexual protist lineage has been described and the existence of such a lineage would stand as an exceptional secondary adaptation to specific environmental conditions (Speijer et al., 2015).

Meiosis genetic toolkit

A meiosis genetic toolkit has been described and can be categorized by the sequence of six general molecular processes characteristic of meiosis: chromosome cohesion, formation of a synaptonemal complex, introduction of double strand breaks (DSBs), chromosomal recombination, crossover resolution, and mismatch repair (Hofstatter et al., 2018; Hofstatter & Lahr, 2019; Malik et al., 2008; Ramesh et al., 2005; Schurko & Logsdon, 2008; Villeneuve & Hillers, 2001) (Table 1; Figure 3). The meiosis genetic toolkit is composed of a consensus of 35 genes of which 11 are meiosis specific according to a recent study (Hofstatter et al., 2018).

The remaining 24 genes are involved in both meiotic and mitotic processes. The meiosis‐specific genes are essential for meiosis to occur and, thus, are expected to be conserved across eukaryotes. This specific set of genes has been employed by numerous studies to identify direct signs of sex among genomes and transcriptomes in various protist clades (Table 2).

It is noteworthy that even cultivated lineages that have a confirmed and extensively described sexual cycle do not harbor all of these meiosis‐specific genes (e.g. in Amoebozoa, Dictyostelium discoideum lacks REC8, HOP1, SPO11, DMC1, MER3, and SPO22 (Hofstatter et al., 2018), while Haptista and Stramenopiles lack SPO22 (Table 2)). These absences indicate either a secondary function loss, a gene replacement or the lack of available genetic data. Cases of gene replacements stand as lineage‐specific innovations in the meiosis‐specific gene‐set and some are well documented. For instance, SPO11 has been lost among Chlorophyta (Archaeplastida) and its function is probably ensured by its paralog SPO11_2 (Tekle et al., 2020). Other lineages lack the complete gene‐set for some molecular processes, such as Ciliophora (Alveolata) which lack all of the proteins for the formation of the synaptonemal complex (HOP1, CH2, and SPO22) (Hofstatter & Lahr, 2019).

Syngamy genetic toolkit

While there is a relatively large body of knowledge concerning the meiotic process, genes related to syngamy are poorly documented in comparison (Brukman et al., 2019, 2022; Pinello et al., 2021; Zhang et al., 2021). Syngamy can be decomposed into four characteristic steps: attraction/recognition, adhesion, plasmogamy, and karyogamy.

Compatible mating cells are attracted and recognized in a strict lineage‐specific way. Multiple mating type determination loci have been identified across protist lineages for Amoebozoa (MATA/B‐C/S genes, Okamoto et al., 2016), unicellular Fungi (MATa/α genes, Hanson & Wolfe, 2017), Chlorophyta (Archaeplastida, MT genes, Hamaji et al., 2008), unicellular Ochrophyta (Stramenopiles, MRP/MRM genes, Russo et al., 2018), and Ciliophora (Alveolata, MTA/B genes, Ma et al., 2020) (Table 1). Compatible gametes adhere to each other either by their membrane and/or by their cilia. In Chlorophyta (Archaeplastida) the interaction between SAG1/SAD1 flagellar receptors on plus/minus cells in Chlamydomonas (Ning et al., 2013) has been described, but also membranous gamete adhesion proteins of the FUS1‐GEX2 family have been reported with homology among other protists (Ning et al., 2013). The FUS1 protein forms a fusion‐enabling complex with MAR1, both being expressed on opposed mating types (Brukman et al., 2022; Pinello et al., 2021). The FUS1‐GEX2 family has also been found among other protists like yeasts (Merlini et al., 2013).

The membrane fusion protein HAP2/GCS1 in the main actor in gamete fusion (Fédry et al., 2017; Zhang et al., 2021). The HAP2/GCS1 fusogen is found in many eukaryotic clades from Excavata and Amoebozoa to Alveolata and has been shown to be essential for fertilization in protists and also multicellular organisms (flowering plants, arthropods, worms, and cnidarians) (Fedry et al., 2018). The multilineage conservation of HAP2/GCS1 indicates an early origin in eukaryotic evolution and its expression is gamete specific (Fedry et al., 2018; Zhang et al., 2021; Table 2). Other less conserved proteins are essential for plasmogamy in a lineage‐specific way, like FIG1 for Chlorophyta (Archaeplastida) (Tekle et al., 2020), FUS2 in yeasts (Merlini et al., 2013), and MAC‐A among Amoebozoa (Araki et al., 2012). Among the rare lineages lacking HAP2/GCS1, the PRM1 gene has been proposed as a candidate mediating membrane fusion in unicellular Fungi (Brukman et al., 2019). A conserved protein family has also been identified governing the process of karyogamy: GEX1‐KAR5 proteins are known to be essential for yeast karyogamy while they have also been shown to play a role in Chlorophyta (Archaeplastida) nuclear fusion (Ning et al., 2013) and have been found in genome and transcriptome data across all major eukaryotic clades (Speijer et al., 2015). Other karyogamy essential genes described in yeasts are KAR3 and KAR4 (Tekle et al., 2020). The abovementioned genes define the basis of a syngamy genetic toolkit, with only two essential and conserved genes to date (HAP2/GCS1 and KAR5/GEX1) (Tables 1 and 2).

In a nutshell, sexual cycle‐specific genes (i.e. meiosis specific and syngamy specific) are encountered in free‐living representatives of all major protist clades (Table 2), with less matches among Discoba and Cryptista, for which there is little or no evidence regarding either meiosis or syngamy available. Overall, the lack of sexual cycle‐specific homologs can be due to a lack of available genetic information (i.e. Cryptista (Figure 2)) and/or high genetic divergence between protist lineages.