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Journal of Experimental Botany, Vol. 70, No. 11 pp. 2951–2964, 2019 doi:10.1093/jxb/erz109 Advance Access Publication 11 March 2019

REVIEW PAPER Did apomixis evolve from sex or was it the other way around?

Emidio Albertini1,*, , Gianni Barcaccia2, , John G. Carman3, and Fulvio Pupilli4, 1 Department of Agricultural, Food and Environmental Sciences, University of Perugia, 06121 Perugia, Italy 2 Laboratory of Genomics, Department of Agronomy, Food, Natural Resources, Animals and the Environment (DAFNAE), University of Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 Padova, 35020 Legnaro, PD, Italy 3 Department of Plants, Soils and Climate, Utah State University, Logan, Utah, USA 4 Institute of Biosciences and Bioresources, Research Division of Perugia, National Research Council (CNR), 06128 Perugia, Italy

* Correspondence: [email protected]

Received 10 October 2018; Editorial decision 25 February 2019; Accepted 25 February 2019

Editor: Sílvia Coimbra, University of Porto, Portugal

Abstract In angiosperms, there are two pathways of reproduction through seeds: sexual, or amphimictic, and asexual, or apo- mictic. The essential feature of apomixis is that an embryo in an ovule is formed autonomously. It may form from a cell of the nucellus or integuments in an otherwise sexual ovule, a process referred to as adventitious embryony. Alternatively, the embryo may form by parthenogenesis from an unreduced egg that forms in an unreduced embryo sac. The latter may form from an ameiotic megasporocyte, in which case it is referred to as diplospory, or from a cell of the nucellus or integument, in which case it is referred to as apospory. Progeny of apomictic plants are generally identical to the mother plant. Apomixis has been seen over the years as either a gain- or loss-of-function over sexu- ality, implying that the latter is the default condition. Here, we consider an additional point of view, that apomixis may be anciently polyphenic with sex and that both reproductive phenisms involve anciently canalized components of complex molecular processes. This polyphenism viewpoint suggests that apomixis fails to occur in obligately sexual eukaryotes because genetic or epigenetic modifications have silenced the primitive sex apomixis switch and/or dis- rupted molecular capacities for apomixis. In eukaryotes where sex and apomixis are clearly polyphenic, apomixis exponentially drives clonal fecundity during reproductively favorable conditions, while stress induces sex for stress- tolerant spore or egg formation. The latter often guarantees species survival during environmentally harsh seasons.

Keywords: Apomeiosis, apomixis, epigenetics, eukaryogenesis, meiosis, origins of sex, parthenogenesis, plant reproduction.

Introduction Apomixis in angiosperms is asexual seed formation (Hand and canalizations (Siegal and Bergman, 2002) of multiple and Koltunow, 2014). However, for eukaryotes in general, apomixis complex molecular processes that have remained conserved is life-cycle renewal through gamete-like cells but without among single-celled and, later, multi-celled species of eukary- sex, i.e. without ploidy reduction (meiosis) or ploidy restitu- otes since single-cell eukaryogenesis (Schurko and Logsdon, tion by syngamy. Both meiosis and syngamy are developmental 2008; Cavalier-Smith, 2010; Bernstein and Bernstein, 2013;

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Hanson et al., 2013; Hörandl and Hadacek, 2013; Speijer et al., innovations of the prokaryotic division phenism (Carman 2015; Bernstein et al., 2018). et al., 2011). Accordingly, apomixis and sex could be anciently Successful apomixis usually silences sexual development polyphenic, with the primary functions of apomixis (repro- within the same ovule. There are reports of twin embryos, one duction without sex) being far older than sex, reaching back formed through apospory and one formed sexually in the same possibly 3.95 Ga ago (Tashiro et al., 2017). If this is correct, sex ovule, but these are rare (Asker and Jerling, 1992). Apomixis and apomixis were probably polyphenic in the last common usually causes germline cells to completely avoid meiosis or, by ancestor of extant eukaryotes. This primitive, single-celled eu- modifications of meiosis, to restore the sporophyte chromo- karyote would have been cyclically apomictic, i.e. it would some number precociously. Either outcome fulfils the ploidy have produced, by apomeiosis, unreduced, non-dormant spores restitution requirement, thus allowing life-cycle renewal to in favourable conditions, and reduced, dormant spores, by mei- proceed without syngamy (parthenogenesis). Apomixis oc- osis, when stressed. This life-cycle pattern occurs today in cyc- curs in thousands of species across all kingdoms of the Eukarya lically apomictic protists, fungi, and animals (Suomalainen et al., (Suomalainen et al., 1987; Bilinski et al., 1989; Hojsgaard et al., 1987; Bilinski et al., 1989), and tendencies toward cyclical apo- 2014). Google Scholar lists ~78 000 papers on apomixis or mixis, with sex usually being induced by unfavorable condi- Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 parthenogenesis, with papers published since 2017 exceeding tions, occur in many angiosperms, including Boechera (Böcher, 4900. Many of these represent original reports of apomixis in 1951; Mateo de Arias, 2015; Gao, 2018), Calamagrostis (Nygren, additional taxa. 1951), Ageratina (Sparvoli, 1960), Limonium (Hjelmqvist and Here, we comment on the origins of apomixis in general; Grazi, 1964), Dichanthium (Knox and Heslop-Harrison, 1963; however, we particularly focus on angiosperms, a youthful Knox, 1967; Saran and de Wet, 1976), Themeda (Evans and group of organisms that are ~130 Ma (million years) old Knox, 1969), Paspalum (Quarin, 1986), Cenchrus (Gounaris (Soltis et al., 2005). Current opinion favours the hypothesis et al., 1991), Ranunculus (Klatt et al., 2016), and Eragrostis that apomixis across the eukaryote kingdoms evolves from sex (Rodrigo et al., 2017). by mutations or epigenetic modifications that deregulate sex (Carman, 1997; Grimanelli, 2012; Hand and Koltunow, 2014; Sex and the precocious ploidy-restitution mechanisms Neiman et al., 2014; Brukhin, 2017). Some authors have sug- of apomixis gested a different possibility, that many eukaryotes have re- tained in their genomes a multi-billion-year-old capacity to The nucellus of the angiosperm ovule is an evolutionary in- reproduce without invoking the eukaryotic innovations of novation of lower plant sporangia. In lower plants, and in meiosis (Carman et al., 2011). Here, we review and present organisms from other major eukaryote groups including further evidence on these divergent hypotheses. Chromista, Protista, and Fungi, sporangial cells generally produce meiotic sporocytes. This is also the case for the angio- Apomixis and sex may be anciently polyphenic sperm anther, where numerous sporocytes differentiate into meiocytes. However, only a single sporogenous nucellar cell Polyphenisms are multiple forms or functions that an organism differentiates into the sporocyte (the megaspore mother cell, can assume in response to environmental signals. The different MMC) in angiospermous ovules. The remaining nucellar cells, phenisms exhibited are encoded by different combinations of while sporangial in origin, generally undergo apoptosis (Herr, genes from the same genome. Environmentally induced meta- 1995). Their sporangial ancestry probably contributes to their bolic signals activate phenism switching upstream, which causes observed competence for apospory. MMCs in angiosperms downstream reprogramming of gene expression. Examples in- generally produce four haploid megaspores by meiosis. One clude environment-driven vegetative to floral transitions in of these undergoes three endomitotic divisions to produce an plants, and the switch in pea aphids and water fleas from apo- 8-nucleate megagametophyte (embryo sac). The other mega- mictic female production in the summer to the production of spores and adjacent nucellar cells undergo programmed cell sexual males and females in response to stress in the autumn death, thereby providing nutrients to the growing embryo sac (Crevillén et al., 2014; Ogawa and Miura, 2014; Bonasio, 2015; (Dwivedi et al., 2014). The fates of nucellar cells, the surviving Crisp et al., 2016; Projecto-Garcia et al., 2017). megaspore, and other megaspores are determined by cell- Cell division versus spore formation is an ancient prokary- cycle regulators and the homeodomain transcription factor otic polyphenism. In metabolically favourable conditions, WUSCHEL (WUS). Transgenically disrupting these pathways prokaryotic cells divide rapidly. In contrast, stress in many pro- allows additional nucellar cells of the ovule to pursue meiosis karyotes slows cell division and induces the spore phenism, and embryo sac formation (Zhao et al., 2017; Cao et al., 2018). wherein cells divide unequally with the larger cell-division This restriction of sporogenous cells is an innovation of the product becoming a stress-resistant spore (McKenney et al., female angiospermous ovule, for example it is not observed 2013; Shimkets, 2013). Sex (haploid spore formation, gamete in fern sporangia (unisexual meioses) or in the angiosperm differentiation, and syngamy) evolved during eukaryogenesis anther. ~1.2 Ga (billion years) ago by co-opting prokaryotic recom- Sporogenous cells can pursue other developmental path- bination and spore-formation processes (Cavalier-Smith, 2010; ways in Protista, Fungi, Chromista, and the lower plants Bernstein et al., 2018). The three main components of apo- and algae of Plantae, as well as sporogenous nucellar cells in mixis (apomeiosis, unreduced gamete differentiation, and par- angiosperms (Fig. 1). For example, in species from 127 angio- thenogenesis) may have evolved simultaneously as mitotic spermous genera, MMCs or their meiotic products die while The mystery of apomixis | 2953 Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021

Fig. 1. Overview of sexual (A) and apomictic (B–H) processes in eukaryotes. Similarities across kingdoms suggest that the life-cycle of primitive eukaryotes, consisting of non-stress-induced mitotic propagation and stress-induced haploid spore formation (sexual), expanded early in eukaryogenesis to include a third phenism, namely non-stress-induced diploid spore formation (apomixis). Red shading indicates premeiotic, meiotic, or early post meiotic phases where ploidy restitution occurs; black bars indicate abrupt developmental termination; the blue-to-green gradients indicate gradual developmental termination; blue arrows indicate gamete or gametophyte development; and large black arrows indicate adventitious embryo development. one or more adjacent nucellar cells per ovule forego apoptosis Kamiya et al., 2011; Takahashi and Mikami, 2017). In gam- and instead produce unreduced embryo sacs (Hojsgaard et al., etophytes of Plantae, unreduced egg formation and egg cell 2014). Eggs in these embryo sacs develop into embryos par- parthenogenesis occur only in angiosperms. However, these thenogenetically, and endosperm forms either pseudogamously processes also occur in brown algae of the kingdom Chromista or autonomously, i.e. with or without fertilization of the (Gall et al., 1996), which acquired some of its kingdom- embryo-sac central cell. Collectively, these processes constitute distinctive features from red algae (Plantae) about 750 Ma ago aposporous apomixis (Fig. 1E). Unreduced aposporous gam- (Cavalier-Smith, 2018). etophytes also form in certain bryophyte (liverwort, hornwort, In species from 85 angiospermous genera, unreduced em- and moss), pteridophyte (fern, horsetail, and lycophyte), and bryo sacs form from ameiotic MMCs (Hojsgaard et al., 2014). red algae species. However, eggs seldom form in the unreduced In Antennaria-type diplospory (gonial apospory), the embryo gametophytes of these lower groups of Plantae. Instead, em- sac forms directly from the MMC (Fig. 1F). In Taraxacum- bryos arise adventitiously from somatic cells of the unreduced type diplospory, the embryo sac forms from one of two gametophytes (Fig. 1H), a process known as apogamy (Asker unreduced spores that arise from the MMC after a mei- and Jerling, 1992; Mogie, 1992; West et al., 1992; Bryan, 2001; otic first-division restitution (Fig. 1B). As with aposporous 2954 | Albertini et al. apomixis, unreduced eggs in diplosporous embryo sacs develop Could the ploidy-restitution mechanisms of sex and into embryos parthenogenetically, and the endosperm forms apomixis be temporal variations of a more general either pseudogamously or autonomously. First-division restitu- restitution signal? tion coupled with parthenogenesis (Fig. 1B) is the most wide- spread form of apomixis among eukaryotes and is the most Both sex and apomixis involve life-cycle renewal from gamete common form in Protista and Fungi (Bilinski et al., 1989), and or gamete-like cells following ploidy restitution. The differ- in Animalia (Suomalainen et al., 1987). ences arise in timing and mechanisms of restitution (Fig. 1). For About 1000 fern species are apomictic (10 %; Mogie, 1992). sex, restitution occurs later, by syngamy, after female and male The most common mechanism involves sporocyte endomitosis, gametes have formed by different meiosis events (Fig. 1A). For wherein the chromatids fail to separate at the centromeres. apomixis, restitution occurs earlier, with parthenogenesis pro- A second (premeiotic) S phase then replicates the two con- viding life-cycle renewal from a gamete-like cell but without joined chromatids of each chromosome, and synapsis and a paternal input. Accordingly, parthenogenetic automixis (sensu crossing-over among the four identical conjoined chromatids Mogie, 1992) in protists, fungi, and animals involves the pre- in each chromosome ensues (Fig. 1C). After two homeotypic cocious fusion of two of four immediate products of a single Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 divisions, four unreduced megaspores form. Because crossing- meiosis followed by (i) differentiation of the ploidy-restored over among and segregation between homologous chromo- cell into an unreduced gamete and (ii) parthenogenesis (Fig. somes does not occur, all spores are clones of the mother plant 1D). Because parthenogenetic automixis in fungi, animals, and (Mogie, 1992). Within leptosporangiate ferns, nearly half of the protists does not include fusion of mature gametes that origin- families contain one or more apomictic taxa (Grusz, 2016). ated from different meiotic events, semantically it is not sex, Apomixis frequencies in ferns correlate well with species di- although it clearly blurs our definitions, i.e. it can generate versity but not with diversification (Liu et al., 2012), which variation through recombination and remodeling of genetic suggests that the apomictic lineages are generally youthful, information, and it often occurs under stress conditions. with most having appeared within 8 Ma (Liu et al., 2012) or 15 Haploid fern gametophytes, which arise from single spores Ma (Tanaka et al., 2014). by mitosis, also present semantic difficulties. Each gametophyte The premeiotic endoreduplication form of apomixis ob- produces eggs, in archegonia, and sperm (genetically identical served in ferns also occurs in angiosperms (Allium odorum- to the eggs), in antheridia. In some ferns, these identical eggs type diplospory), in Allium (Amaryllidaceae) (Kojima and and sperm (from a hermaphroditic gametophyte derived from Nagato, 1997), and possibly in Beta (Amaranthaceae) (Szkutnik, a single spore) fuse to produce a perfectly inbred sporophyte. 2010). As in apospory and other forms of diplospory, the sur- Biologically, this process seems less sex-like than autogamy in viving unreduced Allium odorum-type megaspore under- fungi, animals, and protists, where at least different meiotically goes unreduced embryo sac and egg formation followed by recombined products (from a single meiosis) fuse. Nonetheless, parthenogenesis. we consider it sex because sperm fuse with eggs, even though In some apomictic ferns, Taraxacum-like first-division the specific sperm and egg may have originated from a single meiotic restitutions (Fig. 1B) occur instead of pre-meiotic meiotic product (spore). Other ploidy-restitution mechanisms, restitutions (Fig. 1C). Regardless of the apomeiotic mode including first-division restitution (Fig. 1B), meiotic restitution (Fig. 1B or 1C), clonal embryos in ferns do not form from of ploidy level following a pre-meiotic endoreduplication (Fig. unreduced eggs. Instead, they form apogamously (adven- 1C), and restitution via absence of meiosis, as in apospory and titiously), like those of apomictic red and brown algae, adventitious embryony, are apomictic because sperm do not from somatic cells of unreduced gametophytes (Fig 1H). participate in ploidy restitution. This emphasis on differentiating In fact, egg-producing archegonia, which form in sexu- mechanisms based on whether sperm participate or not (apo ally derived (genetically reduced) fern gametophytes, rarely mixes) may have fostered some incorrect impressions about form in apomeiotically derived unreduced fern gameto- life-cycle renewal, including a perception that sex is supreme, phytes (Mogie, 1992; Grusz, 2016). Interestingly, this ab- i.e. that apomixis and sex must be strongly divergent processes sence of egg-forming archegonia coupled with apogamy evolutionarily or that apomixis somehow involves major evo- in apomictic ferns is consistent with expressions of more lutionary deregulations of sex. ancient gametophyte pathways in lower Plantae, in certain According to the polyphyletic (i.e. multiple ancestral Chromista, and in Protista and Fungi. sources), de novo origins hypothesis, apomixis occurs in plants Adventitious embryony also occurs among species of 148 due to specific mutations (Hand and Koltunow, 2014; Brukhin, angiospermous genera (Hojsgaard et al., 2014). Here, adven- 2017). However, if sex and apomixis are anciently polyphenic, titious embryos generally form from cells of the sporogenous then apomixis or sex might arise in phylogenies because of nucellus or, rarely, the integuments (Fig. 1G) (Nogler, 1984; genomic perturbations that have switched reproduction from Hand and Koltunow, 2014) instead of from unreduced gam- one phenism to the other. Importantly, research targets for etophyte cells as occurs during fern apogamy. Adventitious elucidating apomixis mechanisms will differ substantially de- embryony is more frequently observed in tropical than in tem- pending on the hypothesis one chooses to follow. Based on de perate flora, is better represented among diploid species than novo origins, mutations of genes functioning deep within the other forms of angiospermous apomixis, and is found in several canalized processes of meiosis and syngamy are natural targets. species of Citrus, in mango (Mangifera indica), and in certain In contrast, genes that participate in signal transduction up- orchids (Naumova, 1992). stream of the canalized meiosis and syngamy processes are the The mystery of apomixis | 2955 gene targets based on the polyphenism view, and variations in from a common ancestor, may be inclined to apomixis. As re- timing and location of expression of these genes may regulate markable as the Rosaceae, Poaceae, and Asteraceae are, several the type of apomixis expressed (Fig. 1) (Gao, 2018). In this re- large families may be under-represented, such as the Fabaceae view, we discuss these various views of the evolution and regu- (Leguminosae, ~5 genera) and Orchidaceae (~21 genera) lation of apomixis in terms of recent literature. (Carman, 1997; Hojsgaard et al., 2014). Occurrences of apomixis in angiosperms were comprehensively reviewed by Hojsgaard et al. (2014), and updated lists are available online (http://www. Evolutionary and developmental genetics uni-goettingen.de/en/apomixis+database/423360.html). of apomixis Regarding phylogenetic distributions of apomixis, Carman (1997) reported strong correlations between low chromosome Apomictic biotypes are mainly polyploid, whereas their sexual base numbers for angiospermous genera and the occurrence of counterparts often are diploid (Asker and Jerling, 1992; Pupilli apomixis, and Van Dijk (2009) reported a clustering of apomixis and Barcaccia, 2012), and many appear to be of interspecific at the sub-tribal level in the Poaceae and Asteraceae. The latter hybrid origin (Carman, 1997; Richards, 2003). Because most suggests either a pre-adaptation for apomixis or a common an- Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 hybrids and polyploids are sexual, not apomictic, it remains un- cestry (Van Dijk and Vijverberg, 2006). The basal angiosperms, clear how hybridization and polyploidization cause apomixis Amborelleales, Nymphaeales, and Austrobaileyeales, consist (Osborn et al., 2003; Comai, 2005; Swanson-Wagner et al., of seven families and ~170 species. Embryological data is not 2006). Most angiospermous apomicts are facultative, meaning available for most of these; however, poor or absent pollen for- they set seeds sexually and apomictically with genotype-specific mation in species of Trithuria (Hydatellaceae, Nymphaeales) and sometimes environment-specific frequencies (Asker and suggests apomixis (Remizowa et al., 2008; Iles et al., 2012). Jerling, 1992; Carman et al., 2011). Despite hypothesized dis- Tribe Potentilleae (Rosaceae) provides an excellent example advantages of apomixis, including limited genetic variation and of parallel resurfacing or de novo origins among related genera. accumulation of mutations, apomicts are often highly adapt- Here, sex occurs in Aphanes, Argentina, Comarum, Dasiphora, able and surprisingly stress tolerant. Consequently, questions re- Drymocallis, Farinopsis, Fragaria, Horkeliella, Potentilla, and garding the origins and evolution of sex and apomixis continue Sibbaldia, whereas apomixis is restricted to Potentilla, primarily to confront biologists (Bell 1982; Kirchheimer et al., 2018). with pseudogamous endosperm formation, and to Alchemilla and Aphanes, primarily with autonomous endosperm forma- Parallel and convergent evolution tion. Apomixis in Potentilla is widespread in the Northern Hemisphere, a pattern explained by interspecific hybridizations Classical theory considers apomixis to be a consequence of and introgression as well as repeated intercontinental dispersals sexual silencing or failure rather than a recipe for clonal suc- (Dobes et al., 2015). In general, angiospermous apomicts and cess. Various evidence seems to support the idea of apomixis their sexual relatives are often sympatric, but apomicts typically as a modification of normal sexual development (reviewed by have larger geographic ranges and are better equipped to col- Hand and Koltunow, 2014 and Briggs and Walters, 2016). For onize previously glaciated regions (Bierzychudek, 1985; Bayer, example, apomixis retains non-functional structural aspects of 1997). Many factors contribute to geographic differences, sexual reproduction, while adding functional aspects such as the relative influences of which are species specific (Hörandl, unreduced egg-cell formation and parthenogenesis. In addition, 2006; Kirchheimer et al., 2018). The early evolutionary diver- most angiospermous apomicts require central-cell fertilization, gence (~50 Ma ago) of Potentilleae lineages, characterized by a sexual function, for endosperm formation (pseudogamous pseudogamous and autonomous apomictic seed formation, apomixis; Battaglia, 1963; Nogler, 1984). suggested parallel origins of apomixis (Dobes et al., 2015) due Apomixis occurs in at least 80 families (12%) and 300 genera to repeated evolution of the same phenotype or genotype in (1.8%) of angiosperms (Hojsgaard et al., 2014). However, different populations. ecotype-expansive embryological data (hence reasonably con- Genes responsible for derived phenotypes may be similar clusive) are available for <10% of the 351 000 angiospermous in closely related species (parallel evolution) or different in species, and new families, genera, and species that were pre- distantly related species (convergent evolution). In contrast, viously assumed to be 100% sexual continue to be regularly ancestral traits, such as meiosis, exhibit strong phylogenetic added to apomixis lists (Carman, 1997; Hojsgaard et al., 2014). continuities among related and distantly related taxa. The latter Hence, assuming that unanalysed extant species are sexual is does not appear to be the case for apomixis. Here, major dis- flawed reasoning, and phylogenic analyses of apomixis that do continuities exist among orders, families within orders, genera so are suspect. Nor should we assume that extinct species were within families, and even species within genera (Hojsgaard necessarily sexual. We currently have no idea how pervasive or et al., 2014). Currently, the two main variants of apomixis in ancient apomixis is or how extensive its role has been in the monocots and eudicots, apospory and diplospory, do not appear evolution of new taxa. to have a common ancestor (reviewed by Briggs and Walters, Certain angiospermous families contain many highly apo- 2016). However, as discussed above, it is unknown whether mictic genera. The outstanding examples of this are the apomixis occurred in primitive angiosperms or whether it may Rosaceae, Poaceae (Graminae), and Asteraceae (Compositae). have played a role in speciation. Since apomixis clusters above the genus level in these families, Taking this evidence into account together with results sug- some clades, including closely related species that originated gesting that apomictic lineages could be young in ferns (e.g. 2956 | Albertini et al.

<8 Ma, Liu et al., 2012; <15 Ma, Tanaka et al., 2014) has led 2012; Barcaccia and Albertini, 2013; Conner et al., 2015, geneticists to assume that apomixis is highly derived and must 2017). Recently, artificial apomixis systems have been en- have arisen polyphyletically hundreds of times (Carman, 1997; gineering into rice (Khanday et al., 2019; Wang et al., 2019, Briggs and Walters, 2016). Some authors consider this conclu- see below). However, attempts to engineer a natural form of sion as unsettling because de novo evolution of apomixis from apomeiosis into plants have failed, which suggests that the sex might require highly specific interventions in two con- genetic control of apomixis is more complicated than pre- served and molecularly complex canalized processes, i.e. mei- viously perceived. An example of possible complications in- osis and syngamy. It seems probable that evolution of apomixis volves SPOROCYTELESS (SPL)/NOZZLE (NZZ). Loss of from a sexual context would require a combination of muta- SPL/NZZ function abolishes sporogenesis in both male and tions or epigenetic disturbances that would be quite difficult to female organs (Yang et al., 1999; Liu et al., 2009). If SPL/NZZ recreate independently and repeatedly throughout phylogeny. (or other genes) are required for both sex and apomixis, then Moreover, that these putative mutations should occur simul- disrupting them might also deleteriously disrupt apomeiosis taneously such that an organism remains viable during the pro- and parthenogenesis. cess of apomixis evolution is difficult to explain (Mogie, 1992; In recent decades, scientists have selected apomixis candidate Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 Neiman et al., 2014). However, it is possible that apomixis is genes from among the deep molecular channels controlling the governed by a single mutation that causes functional alterations functioning of meiosis and syngamy with the hope that muta- in both processes (due to actions at different targets and in tions of such genes will mimic naturally occurring apomixis- association with different partners). This possibility cannot be inducing mutations. This approach has met with limited success dismissed in species where the trait appears to be controlled (Pupilli and Barcaccia, 2012; Barcaccia and Albertini, 2013). In by a single locus. Alternatively, molecular cascades involved contrast, expressing a BABY BOOM (BBM) gene (APETALA in apomixis might show branching only after a considerable 2/ETHYLENE RESPONSE FACTOR, AP2/ERF family number of intracellular reactions, which could regenerate the of transcription factors), cloned from an apomictic grass, in same phenotype by affecting different genes. Biologists have eggs of sexual cereals has recently been shown to induce par- wrestled with this paradox and have hypothesized plausible thenogenesis (Conner et al., 2015, 2017). Several members sequences of events whereby apomixis may have evolved (de- of this gene family induce embryogenesis in sporophytic or scribed below). In contrast, if apomixis and sex are anciently gametophytic tissues of angiosperms and ferns (Boutilier et al., polyphenic, then periods of deep evolution could occur during 2002; Bui et al., 2017; Horstman et al., 2017). Some ERFs par- which apomixis remains silent. ticipate in stress signalling (Müller and Munné-Bosch, 2015) Meiosis and syngamy probably evolved as DNA repair pro- by up-regulating α-amylase genes, which leads to glucose cesses and are induced by oxidative stress (Cavalier-Smith, signalling and the activation of TARGET OF RAPAMYCIN 2010; Hörandl and Hadacek, 2013; Bernstein et al., 2018). That (TOR). Associated oxidative bursts initiate ROS signalling and sexual reproduction dominates eukaryote evolution may re- the production of ROS scavengers (Qin et al., 2017; Considine, flect the fact that oxidative stress is required not only for DNA 2018). Hence, the BBM gene used by Conner et al. (2015, repair, but for life in general (Mittler and Blumwald, 2015). 2017) may have affected signalling cascades upstream of syn- In this respect, apomeiosis might only occur in response to gamy rather than syngamy itself. A wild-type BBM gene (not shifts in redox homeostasis that increase reactive oxygen spe- from an apomict) also induces parthenogenesis when expressed cies (ROS-)scavenging capabilities (Gao, 2018; Sherwood, in egg cells (Khanday et al., 2019), which suggests that meta- 2018). Such shifts in metabolic homeostasis could occur as a bolic factors regulate apomixis rather than apomixis-specific consequence of hybridization and polyploidization (Kirk et al., mutations. 2005). Accordingly, phylogenetic discontinuities, with apo- Genetic studies of the last century indicated that apomixis is mixis occasionally surfacing in terminal nodes, may occur in simply inherited (Grossniklaus et al., 2001; Ozias-Akins, 2006; response to metabolically induced epigenetic changes rather Whitton et al., 2008; Pupilli and Barcaccia, 2012; Hand and than mutation-derived changes. Koltunow, 2014), and multiple mutations have been identi- fied that affect individual components of apomixis (Curtis and The genetic control of apomixis Grossniklaus, 2007; Ravi et al., 2008). Likewise, the three com- ponents of apomixis, namely apomeiosis, parthenogenesis, and Genetic recombination and fusion of divergent gametes en- functional endosperm formation (without a 3:2 endosperm ables diversification and adaptation, while clonal seed for- to embryo genome ratio), have been uncoupled experimen- mation (apomixis) perpetuates highly successful genotypes tally. This has been documented in Taraxacum (Van Dijk et al., without the interference of genetic reshuffling. Facultative 1999; Van Dijk, 2003), Erigeron (Noyes and Rieseberg, 2000), apomixis provides the best of both worlds, for example highly Poa (Barcaccia et al., 2000; Albertini et al., 2001), Hypericum successful genotypes may persist for hundreds of years with (Barcaccia et al., 2006; Schallau et al., 2010), Cenchrus (Conner facultative apomixis producing new genotypes to accommo- et al., 2013), and Hieracium (Catanach et al., 2006; Henderson date changing habitats. In the face of changing environments, et al., 2017). These findings suggest that apomixis-conferring successful sexual recombinations might only rarely be required. loci are complex with perhaps multiple genes affecting each Scientists have studied genes controlling key steps in apo- pathway. They also suggest that mutations outside of the ca- mixis and have engineered plants with candidate genes that nonical apomixis loci can affect the penetrance of apomixis induce apomixis-like phenotypes (Pupilli and Barcaccia, elements. Accordingly, devising a universal model for the The mystery of apomixis | 2957 origins and expression of apomixis may be unrealistic (Briggs studied by comparative mapping. A single, complex, dominant and Walters, 2016). locus controls apomixis in both systems and is characterized by strong repression of recombination and other features of heterochromatin, including accumulation of transposable Evolution of the apomixis locus within the elements (TEs) and DNA methylation. Although no relevant Gramineae large-scale collinearity/synteny between the apomixis locus Despite frequent events of genomic repatterning and gene and reference grass genomes has been detected in Pennisetum losses, which have caused considerable divergence in genome (Conner et al., 2008), comparative mapping in Paspalum has size in the grass family (Bennetzen and Wang, 2018), genes shown that the apomixis locus is macro-syntenic with the have tended to maintain their position in chromosomes telomeric part of rice chromosome 12 (Pupilli et al., 2001, with strict conservation of gene order (collinearity) or gene 2004; Martinez et al., 2003; Hojsgaard et al., 2011). Studies at co-localization (synteny) (Wang et al., 2015 and references the sequence level also confirm this (Calderini et al., 2006), therein). Since grass genomes are largely syntenic and apo- although the sequence is often interrupted by gene loss and mixis occurs throughout the grass family, several authors have migration. Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 thought that the same set of genes may control apomixis in Recently, Galla et al. (2019) have extended our know- grasses (Kellogg, 2015). However, while the regions control- ledge beyond rice and found that the apomixis locus is also ling apomixis are similar within genera (Pupilli et al., 2004; syntenic with the telomeric regions of chromosomes 8, 3, Akiyama et al., 2011), they can be highly divergent between and 4 of Sorghum, Setaria, and Brachipodium, respectively, and genera (Ozias-Akins, 2006). This is consistent with the obser- in a more centromeric position on chromosome 1 of maize vation that apomixis has originated or resurfaced many times (Fig. 2). Regions of homology between Sorghum and Setaria independently in the grass family (Ozias-Akins et al., 2003) are larger than for rice due to additional rice genes that mi- and has spread among several species of the same taxa by hy- grated to this region from other locations in the genome. bridization (Worthington et al., 2016). Rearrangements have occurred frequently in this region The apomixis loci of Pennisetum squamulatum (Sapkota et al., during the evolution of grass genomes (Devos et al., 2017; 2016) and Paspalum species (Ortiz et al., 2013) are the best http://www.gramene.org/).

Fig. 2. Alignment of the apomixis locus of Paspalum simplex with its homoeologous counterparts in five reference grass genomes. The two apomixis- linked genes of Paspalum shown here identify the two more external marker genes for which clear homologs were identified in all the reference genomes considered. *apo locus is not to scale. 2958 | Albertini et al.

Case study: what triggered apomictic reproduction in number of characteristics with Y chromosomes, including re- Paspalum during its evolution? pression of recombination, presence of TEs, and gene degen- eration (Pupilli and Barcaccia, 2012). Y chromosomes evolved Apomixis in Paspalum, as in many taxa, is strictly associated with through an initial suppression of recombination in regions of polyploidy, and apomictic species of Paspalum often include autosomal chromosomes that contained sex-expression genes. obligate diploid sexual and polyploid apomictic cytotypes. Further stepwise expansion of recombinational suppression This defines an agamic complex in the classical sense (Stebbins occurred across the chromosome, producing in most cases het- 1950). The most frequent mechanism of polyploidization is the eromorphic Y chromosomes. Consequently, Y chromosomes ‘triploid bridge’ (Ramsey and Schemske 1998), which involves silence ‘female’ expression through the action of degenerated fusion of a reduced gamete, egg, or sperm with an unreduced genes, and they simultaneously encode development of male- gamete (Quarin et al., 1982; Norrmann et al., 1989). This BIII specific organs that function in response to master regulator hybridization gives rise to a triploid (2n+n; 2x+x=3x). Fusion genes (Bergero and Charlesworth, 2009). The apomixis locus of an unreduced gamete of the BIII hybrid with a reduced of Paspalum shares commonalities with operon-like gene clus- gamete of a diploid can then generate a tetraploid (2n+n; ters that in plants control complex traits. These include their Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 3x+x=4x). Alternatively, fusion of an unreduced gamete from a subtelomeric origins and coordinated expression (Boycheva diploid with a reduced gamete of a tetraploid can also produce et al., 2014). Such gene clusters evolved de novo by initial a new tetraploid (2x+2x=4x; Quarin, 1992; Siena et al., 2008). gene duplication followed by neo- or sub-functionalization Hybridization and ploidy state transitions initially cause a ‘gen- and genome rearrangements. Several apomixis-linked genes omic shock’, which correlates with a temporary expression produce sense or antisense transcripts in reproductively of sexuality, as reported in Eragostis curvula (Zappacosta et al., committed cell lineages, which suggests that they might be 2014). However, in time, re-methylation leads to the emer- co-expressed (Galla et al., 2019). For example, the apomixis- gence of a new epigenetic landscape (Zappacosta et al., 2014). linked PsORC3 allele represses expression of its sexual coun- This re-methylation, possibly associated with the intranuclear terpart (Siena et al., 2016). The female phenotype is thought to sensing of an increased ploidy level, or derived from sequence be the default state in some dioecious plants (Janousek et al., variation generated during a previous genomic shock, in- 1996). Likewise, the sexual phenotype is considered by some to duces apomixis, which overcomes gamete sterility. The poly- be the default state in apomixis systems (Koltunow et al., 2011). ploid that is subsequently generated can either discard or retain Here, operon-like superloci are thought to suppress sex and apomixis (Hojsgaard, 2018). Triploids that reproduce by apo- induce apomixis. Podio et al. (2014) showed that the partheno- mixis occasionally occur among diploid P. simplex populations genesis program requires DNA methylation at the apomixis (Urbani et al., 2002). As an important apomictic forage crop, locus, which silences the syngamy requirement. large breeding populations of Paspalum are available to study the evolution of apomixis under recurrent cycles of self-pollin- ation and outcrossing. Apomixis was not observed in a large Epigenetic control of sexuality over synthetic population of Paspalum developed by intercrossing apomixis, or the other way around? sexual hybrids obtained by hybridizing naturally occurring apomictic tetraploids with experimentally derived sexual tetra- It is widely recognized that apomixis is genetically controlled, ploids (Zilli et al., 2018). In addition, full apomixis was not but despite this hypotheses on its origins and inheritance are observed among 20 tetraploids obtained by tissue culture, al- still contradictory and elusive (Ozias-Akins and van Dijk, 2007; though ovules containing aposporic embryo sacs were occa- León-Martínez and Vielle-Calzada, 2019). sionally observed (Acuña et al., 2007). The sexual genotypes Deregulations of key developmental steps in sexual processes used in this study did not contain apomixis-linked markers. are thought to cause apomixis, and supporters of this hypoth- Hence, at the population level, apomixis in Paspalum appears esis justify it based on the coexistence of sex and apomixis to be controlled by a heritable locus, whereas the occasional in the same individual (Koltunow and Grossniklaus, 2003). In occurrence of aposporic embryo sacs in natural or induced contrast, Hojsgaard (2018) suggests that epigenomic shocks sexuals is probably a trait with very low heritability. due to stress, interspecific hybridization, and ploidy-state tran- Apomixis in Paspalum may have arisen by the grouping sitions are the cause of apomixis. While there are several types of multiple sexual development genes in the same genomic of apomixis, there is only one type of sexuality, from which region during speciation followed by polyploidization- apomixis types presumably evolved. In contrast, the coexist- induced rearrangements, such as inversions, that produced ence of apospory and diplospory in some species suggests a recombinationally isolated chromosomal segments that accu- closer relationship between apomixis types, and this could be mulated high genetic load (Calderini et al., 2006). Gene de- due to gene duplications and mutations accumulating inde- regulation (Polegri et al., 2010) followed by the expression of pendently in the same ecotypes. Garcia-Aguilar et al. (2010) constitutively expressed pseudo-genes, as in the case of the found that deregulation of DNA methylation in reproductive apomixis-linked PsORC3, then silence their sex-specific coun- cells induces apomeiosis-like phenotypes, suggesting that spe- terparts via a sense–antisense mechanism (Siena et al., 2016). cialization of a DNA methylation pathway acts upon germline The apomixis locus of Paspalum, which is similar to that or germline-associated cells. Gao (2018) observed a high- of Pennisetum (Ozias-Akins et al., 1998), Brachiaria (Pessino frequency of conversion from Taraxacum-type diplospory (first et al., 1998), and Tripsacum (Grimanelli et al., 1998), shares a meiotic division restitution) to normal meiotic tetrad formation The mystery of apomixis | 2959 when Boechera pistils were exposed to stress treatments within temporal or spatial variations in genetic- and environment- 48 h of when apomeiosis normally occurred. A variety of stress regulated metabolic signalling. The nature of the epigenetic treatments induced these conversions, including carbohy- regulation that orchestrates these phenism transitions requires drate starvation, osmotic stress, exposure to H2O2, and inhib- further investigation. However, that sex and apomixis evolved ition of brassinosteroid synthesis. In addition, Antennaria-type during eukaryogenesis from an apomixis-like background is diplospory (gonial apospory or mitotic diplospory, i.e. no mei- becoming an attractive hypothesis for explaining these findings. otic onset) occurred when DNA methylation was suppressed Changes in ploidy level, interspecific hybridization, and/ prior to MMC formation. These conversions, from apomixis or intraspecific hybridization often precede transitions from to sex and from one apomeiotic type to another, are evidence a sexual to an asexual mating system. Following such gen- that the metabolic status of ovules (and the genes that affect omic events, epigenetic mechanisms controlling TE activity this status) regulate the developmental sex/apomixis decision, can be disrupted, which can lead to a rapid increase in TE and that the type of apomixis expressed is a function of the copy number and/or activity (Bardil et al., 2015). During this temporal and spatial expression of perhaps a single apomixis- process, a subset of elements might escape epigenetic con- conferring signal. trol. Over evolutionary time, selection against asexual lineages Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 Many of the recombination processes of eukaryotic sex, with high TE proliferation rates could favor lineages where which repair ROS-damaged DNA, as well as the epigenet- deleterious TEs are controlled and silenced, thus preserving a ically regulated polyphenic processes of stress-tolerant spore functional genomic architecture. Accordingly, the dynamics of formation were co-opted from prokaryotic ancestors during TE accumulation and extinction in asexual lineages appears to eukaryogenesis (Cavalier-Smith, 2010). A potent ROS-driven represent a specific and dynamic evolutionary process. epigenetic change involves the alteration of global DNA Chaudhury and Peacock (1993) hypothesized that genes CpG methylation patterns. In particular, ROS promote global isolated in model species such as Arabidopsis thaliana would CpG hypo-methylation in DNA, which is associated with in- be important for the study of apomixis, and this led to the creased cellular apoptosis (Fan et al., 2001; Wu and Ni, 2015; identification of several genes that, when mutated, resemble Notley et al., 2017). Moreover, the relationship between or restore components of apomixis (Barcaccia and Albertini, ROS, epimutations, and cell fate has been widely studied in 2013). Today, candidate genes involved in epigenetic switching humans, where ROS has been associated with global CpG between apomixis and sex are being investigated using two hypomethylation of DNA and can facilitate the aberrant his- approaches: identifying epigenetic differences between apo- tone-3 (H3) phosphorylation that leads to apoptotic elimin- mictic and sexual genotypes in nature, and inducing epigenetic ation of cells (Tikoo et al., 2001; Wu and Ni, 2015). In such changes in sexual species that produce phenotypes that mimic cells, oxidative imbalance can alter epigenetic chromatin modi- particular steps of apomixis. fications, leading to cellular catastrophe (Chatterjee and Law, Several studies have been carried out on apomictic species 2018). with the aim of identify epigenetic changes related to the fate In line with its evolutionary origins (Cavalier-Smith, 2010), of the reproductive program. Some of the first observations the regulation of oxidative stress has been proposed as central involved species for which the apomixis locus resided within to sex/apomixis switching in facultatively and cyclically apo- heterochromatin that was found to be enriched in retrotrans- mictic eukaryotes (Carman et al., 2011; Hörandl and Hadacek, posons and repetitive DNA (Calderini et al., 2006; Conner 2013). Several lines of evidence suggest that transitions during et al., 2008). Such conditions are correlated with the methy- reproduction and early seed development are epigenetically lation status of DNA. For example, comparative analyses of regulated by dynamic changes in chromatin state (Huanca- small-RNA, histone modifications, and cytosine methylation Mamani et al., 2005; Xiao et al., 2006; Baroux et al., 2007; Curtis in apomictic and sexual Cenchrus ciliaris (Kumar, 2017) led and Grossniklaus, 2008; Olmedo-Monfil et al., 2010). There is to the identification of different classes of retrotransposons. also growing support for this from studies with sexual plants Among 19 tested, six were preferentially active in apomictic where mutations or pharmacological treatments alter epigen- plants, and one of these was hyper-methylated in sexual plants etic pathways, leading to apomixis-like phenotypes. In this re- (95% versus 35% 5-methyl cytosines in sexual and apomictic spect, Gao (2018) observed a high frequency of conversions plants, respectively). Sexual and apomictic E. curvula genotypes from meiotic tetrad formation in obligately sexual Arabidopsis also produce differentially expressed TE-related sequences thaliana, Boechera stricta, and Vigna unguiculata to Antennaria- (Romero et al., 2016), and similar differences have been noted type diplospory, Taraxacum-type diplospory and/or Hieracium- for apomictic plants obtained by chromosome doubling type apospory when pistils were treated with combinations of (Zappacosta et al., 2014). Sexual genotypes express a Copia-10 5-azacytidine, brassinosteroid, antioxidants, and/or sugars. like element, which the apomictic genotypes do not express. In the naturally diplosporous Eragrostis curvula, Rodrigo This suggests a selective activation of TE between sexual and et al. (2017) observed stress-induced increases in sexual pro- apomictic genotypes. These findings support the notion that cesses in facultative genotypes, but did not observe apomictic TEs and the epigenetic machinery play key roles in fostering processes originating in sexual genotypes of the same species phenotypic and biological innovations during major ecological (J.M. Rodrigo, Universidad Nacional del Sur, Argentina, per- transitions (Fedoroff, 2012), and such mechanisms may facilitate sonal communication). These findings are further evidence transitions from apomixis to sexuality in facultative apomicts. that sex and apomixis, as well as different apomeiosis types, In this respect, Leblanc et al. (2009) demonstrated that the apo- may be polyphenisms of each other and may occur due to mixis locus in maize–Tripsacum hybrids is not inherited in a 2960 | Albertini et al. functional state (i.e. able to induce apomixis) when transmitted A. thaliana and maize (Huanca-Mamani et al., 2005; Xiao et al., through a reduced female gamete. However, it is functional 2006; Baroux et al., 2007; Curtis and Grossniklaus, 2008; Garcia- when transmitted via male meiosis. These studies demonstrated Aguilar et al., 2010; Olmedo-Monfil et al., 2010, Koltunow a parent-of-origin effect. et al., 2011). In particular, nucellar cells in ago9 mutants fail to Interspecific hybridization and allopolyploidy have been undergo programmed cell death but instead initiat develop- proposed as sources of ‘genomic shocks’ that may cause pheno- mental processes reminiscent of apospory. While the apospory- typic alterations through new genetic or epigenetic inter- like structures fail to develop further, evidence was provided actions derived from the activity of divergent genomes or that epigenome-level regulation is important in directing fe- dosage changes in factors that regulate development (Beck male germ-cell development (Olmedo-Monfil et al., 2010). et al., 2012). Several authors have suggested that apomixis in as- In maize, the loss-of-function mutant ago104 (a homolog of sociation with polyploidy could have evolved because together AGO9) produces apomixis-like phenotypes that gave rise to they can mask and perpetuate uneven ploidy levels, aneuploidy, 70% functional unreduced female gametes (Singh et al., 2011). and deleterious mutations. Residual sexuality in facultative Garcia-Aguilar et al. (2010) have shown that inactivation apomicts could be triggered by environmental cues. In this re- of the maize DNA methyltransferases dmt102 and dmt103, Downloaded from https://academic.oup.com/jxb/article/70/11/2951/5373086 by guest on 02 October 2021 spect, facultatively apomictic Boechera switch to meiosis under which are expressed in sexual ovules, results in apomixis-like drought or heat stress (Mateo de Arias, 2015). This is similar to phenotypes, including multiple unreduced embryo sacs. The what was seen by Klatt et al. (2016) in a facultatively apomict authors suggested that DMT102 activity in the maize ovule Ranunculus under extended photoperiods, by Gao (2018) with negatively regulates the transcriptional competence of chro- pharmacologically imposed stress treatments, and by Rodrigo matin in the archesporial tissue, a condition that might be es- et al. (2017) in flowers of stressed E. curvula. In the latter study sential for proper sexual development. Collectively, the mutant it was found that the percentage of sexual embryo sacs in- phenotypes of ago9 in Arabidopsis and dmt103 in maize sug- creased significantly from 1.8–4% in normally watered plants gest that the regulation of DNA methylation in precursor cells to 14.4–22% in plants under water-stress conditions. Removal of female gametes plays a role in differentiating between apo- of stress restored methylation and apomixis in E. curvula. mictic and sexual reproduction. Shah et al. (2016) compared seedlings of an obligate triploid The recent development of ‘artificial’ apomictic rice (Boechera c.f. gunnisoniana) and a sexual line (Boechera stricta) and (Khanday et al., 2019, Wang et al., 2019) provides evidence for hypothesized that osmotic stress and DNA methylation could both evolutionary models, i.e. apomeiosis induced by muta- be involved in gene regulation during apomictic seedling de- tions of meiosis-specific genes and parthenogenesis induced velopment. Consistent with methylation being involved in re- by modulations of wild-type gene expression. Additional re- productive fate, significant changes in genomic methylation search is required to determine whether the artificially cre- patterns occur during the formation of triploid diplosporous ated apomictic rice lines resemble the origins of apomixis in dandelions produced from a diploid sexual mother fertilized nature. Perhaps a combination of both phenomena is respon- by a tetraploid pollen donor (Verhoeven et al., 2010a, 2010b). sible. Concomitant circumstances may have induced mutations In newly tetraploidized E. curvula genotypes, Zappacosta for some aspects of apomictic development and modulated ex- et al. (2014) observed reduced embryo sac formation; how- pression of wild-type genes for other aspects, with apomictic ever, four years later, these perennial plants were producing reproduction emerging from the ‘chaos’. unreduced embryo sacs in 85–90% of their ovules. The authors proposed that genomic shock due to the stress of in vitro cul- ture and polyploidization caused extensive demethylation, and Concluding remarks that this demethylation favored sexual reproduction. During the subsequent four years, remethylation occurred, and apo- The quest to develop apomixis technology for agriculture mixis expression re-emerged. has pushed biologists to acquire deeper understanding of the Podio et al. (2014) reported a decrease of parthenogenesis mechanisms of the sexual and apomicticprocesses, knowledge in a natural apomictic Paspalum simplex genotype after general that appears to be a prerequisite for success. In this review, we artificial genome demethylation. Koltunow et al. (2011) dem- have considered the possibility that apomixis is a highly con- onstrated in H. pilosella that sexual cues enabling meiotic served but evolutionarily suppressed alternative to sex, that tetrad formation in ovules are required for the formation of apomixis and sex are anciently polyphenic, and that the switch aposporous initial (AI) cells. By inference, these cues promote between apomixis and sex is controlled by upstream epigenetic the activity of the dominant LOSS OF APOSPORY (LOA) processes that are environmentally triggered. locus, which functions to stimulate AI cell formation. It was The evidence presented here shows that apomixis involves hypothesized that the sexual cues are hormonal (Koltunow pathways of gene expression that are controlled epigenetically. et al., 2011) or epigenetic. It remains to be determined whether A clear explanation of this control currently does not exist, but epigenetic pathways contribute to the promotion of LOA progress toward a molecular understanding of apomixis may have function or whether such epigenetic elements are the func- finally turned a corner. Genes important to sexual reproduction tional determinants at the LOA locus in Hieracium (Koltunow in some cases now appear to play roles in apomixis as well. Selva et al., 2011). et al. (2017) have identified homologs of AGO genes in the floral Epigenetic pathways appear to be involved in restricting transcriptome of Eragrostis curvula that reveal an unexpected ec- sexual female gametophyte formation to a single cell in both topic pattern during germ-line differentiation in apomicts. The mystery of apomixis | 2961

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