Sexual and apomictic development in Hieracium

 Most members of the genus Hieracium are apomictic and set seed without fertilization, but sexual forms also exist. A cytological study was conducted on an apomictic accession of H. aurantiacum (A3.4) and also H. piloselloides (D3) to precisely define the cellular basis for apomixis. The apomictic events were compared with the sexual events in a self-incompatible isolate of H. pilosella (P4). All plants were maintained as vegetatively propagated lines each derived from a single plant. Sexual P4 exhibited characteristic events of polygonum-type embryo sac formation, showed no latent apomitic tendencies, and depended upon fertilization to set seed. In contrast, D3 and A3.4 were autonomous aposporous apomicts, forming both embryo and endosperm spontaneously inside an unreduced embryo sac. The two apomicts exhibited distinct mechanisms, but variation was also observed within each apomictic line. Seeds from apomicts often contained more than one embryo. A degree of developmental instability was also observed amongst germinated seedlings and included variation in meristem and cotyledon number, altered phyllotaxis, callus formation, and seedling fusion. In most cases abnormal seedlings developed into normal plants. Such phenomena were not observed following germination of hybrid seeds derived from crosses between sexual P4 and the apomictic plants. The three plants can now be used in inheritance studies and also to investigate the molecular mechanisms controlling apomixis. Received: 11 February 1998 / Revision accepted: 23 July 1998     

Sexual and apomictic reproduction in Hieracium subgenus pilosella are closely interrelated developmental pathways

Seed formation in flowering plants requires meiosis of the megaspore mother cell (MMC) inside the ovule, selection of a megaspore that undergoes mitosis to form an embryo sac, and double fertilization to initiate embryo and endosperm formation. During apomixis, or asexual seed formation, in Hieracium ovules, a somatic aposporous initial (AI) cell divides to form a structurally variable aposporous embryo sac and embryo. This entire process, including endosperm development, is fertilization independent. Introduction of reproductive tissue marker genes into sexual and apomictic Hieracium showed that AI cells do not express a MMC marker. Spatial and temporal gene expression patterns of other introduced genes were conserved commencing with the first nuclear division of the AI cell in apomicts and the mitotic initiation of embryo sac formation in sexual plants. Conservation in expression patterns also occurred during embryo and endosperm development, indicating that sexuality and apomixis are interrelated pathways that share regulatory components. The induction of a modified sexual reproduction program in AI cells may enable the manifestation of apomixis in HIERACIUM:     

Dynamics of callose deposition and beta-1,3-glucanase expression during reproductive events in sexual and apomictic Hieracium

Callose accumulates in the walls of cells undergoing megasporogenesis during embryo sac formation in angiosperm ovules. Deficiencies in callose deposition have been observed in apomictic plants and causal linkages between altered callose deposition and apomictic initiation proposed. In apomictic Hieracium, embryo sacs initiate by sexual and apomictic processes within an ovule, but sexual development terminates in successful apomicts. Callose deposition and the events that lead to sexual termination were examined in different Hieracium apomicts that form initials pre- and post-meiosis. In apomictic plants, callose was not detected in initial cell walls and deficiencies in callose deposition were not observed in cells undergoing megasporogenesis. Multiple initial formation pre-meiosis resulted in physical distortion of cells undergoing megasporogenesis, persistence of callose and termination of the sexual pathway. In apomictic plants, callose persistence did not correlate with altered spatial or temporal expression of a β-1,3-glucanase gene (HpGluc) encoding a putative callose-degrading enzyme. Expression analysis indicated HpGluc might function during ovule growth and embryo sac expansion in addition to callose dissolution in sexual and apomictic plants. Initial formation pre-meiosis might therefore limit the access of HpGluc protein to callose substrate while the expansion of aposporous embryo sacs is promoted. Callose deposition and dissolution during megasporogenesis were unaffected when initials formed post-meiosis, indicating other events cause sexual termination. Apomixis in Hieracium is not caused by changes in callose distribution but by events that lead to initial cell formation. The timing of initial formation can in turn influence callose dissolution. Received: 18 April 2000 / Accepted: 10 July 2000     

Sexual reproduction is the default mode in apomictic Hieracium subgenus Pilosella, in which two dominant loci function to enable apomixis

Asexual seed formation, or apomixis, in the Hieracium subgenus Pilosella is controlled by two dominant independent genetic loci, LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP). We examined apomixis mutants that had lost function in one or both loci to establish their developmental roles during seed formation. In apomicts, sexual reproduction is initiated first. Somatic aposporous initial (AI) cells differentiate near meiotic cells, and the sexual pathway is terminated as AI cells undergo mitotic embryo sac formation. Seed initiation is fertilization-independent. Using a partially penetrant cytotoxic reporter to inhibit meioisis, we showed that developmental events leading to the completion of meiotic tetrad formation are required for AI cell formation. Sexual initiation may therefore stimulate activity of the LOA locus, which was found to be required for AI cell formation and subsequent suppression of the sexual pathway. AI cells undergo nuclear division to form embryo sacs, in which LOP functions gametophytically to stimulate fertilization-independent embryo and endosperm formation. Loss of function in either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in total reversion to sexual reproduction. Therefore, in these apomicts, sexual reproduction is the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode genes essential for sexual reproduction, but may function to recruit the sexual machinery at specific time points to enable apomixis.     

Is supernumerary chromatin involved in gametophytic apomixis of polyploid plants?

Gametophytic apomixis, or unreduced embryo sac development that results in asexual reproduction through seeds, occurs in several families of angiosperms and must be polyphyletic in origin. The molecular mechanisms underlying gametophytic apomixis have not been discovered and are the subject of intense investigation. A common feature of almost all apomicts is their polyploid nature. From genetic mapping studies in both monocots and dicots, there is low genetic recombination associated with a single (rarely two), dominant locus for either aposporous or diplosporous embryo sac formation. In Pennisetum squamulatum and Cenchrus ciliaris, some DNA sequences mapping to the apospory locus are unique to apomictic genotypes and apparently hemizygous. This sequence divergence at the apomixis locus could be a consequence of genome rearrangements and isolation from genetic recombination, both of which may have contributed to the definition of a chromosomal region as supernumerary. The possible involvement of supernumerary chromatin, formed as a result of interspecific hybridization, in the origin of apomixis, is explored here. Received: 26 October 2000 / Revision accepted: 5 April 2001     

Evolution of apomixis loci in Pilosella and Hieracium (Asteraceae) inferred from the conservation of apomixis-linked markers in natural and experimental populations

The Hieracium and Pilosella (Lactuceae, Asteraceae) genera of closely related hawkweeds contain species with two different modes of gametophytic apomixis (asexual seed formation). Both genera contain polyploid species, and in wild populations, sexual and apomictic species co-exist. Apomixis is known to co-exist with sexuality in apomictic Pilosella individuals, however, apomictic Hieracium have been regarded as obligate apomicts. Here, a developmental analysis of apomixis within 16 Hieracium species revealed meiosis and megaspore tetrad formation in 1 to 7% of ovules, for the first time indicating residual sexuality in this genus. Molecular markers linked to the two independent, dominant loci LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) controlling apomixis in Pilosella piloselloides subsp. praealta were screened across 20 phenotyped Hieracium individuals from natural populations, and 65 phenotyped Pilosella individuals from natural and experimental cross populations, to examine their conservation, inheritance and association with reproductive modes. All of the tested LOA and LOP-linked markers were absent in the 20 Hieracium samples irrespective of their reproductive mode. Within Pilosella, LOA and LOP-linked markers were essentially absent within the sexual plants, although they were not conserved in all apomictic individuals. Both loci appeared to be inherited independently, and evidence for additional genetic factors influencing quantitative expression of LOA and LOP was obtained. Collectively, these data suggest independent evolution of apomixis in Hieracium and Pilosella and are discussed with respect to current knowledge of the evolution of apomixis.     

A DEFICIENS homologue is down-regulated during apomictic initiation in ovules of Hieracium

 Hieracium is a member of the Asteraceae family, and contains sexual species in addition to apomictic species that reproduce by apospory and produce seed without fertilization. A homologue of the floral organ-identity gene DEFICIENS (DEF) was isolated from an apomictic line of Hieracium piloselloides (Vill.) following differential display between mature ovules and those initiating autonomous embryogenesis. The gene termed HPDEF has 93% amino acid identity with GDEF2, a DEF homologue isolated from Gerbera hybrida (D. Yu et al., 1999, Plant J. 17: 51–62), another member of the Asteraceae. In-situ analysis showed that early in floral development HPDEF is expressed in stamen and petal primordia, indicating expected B-function activity, according to the ABC model of floral organ identity (J. L. Bowman et al., 1991, Development 112: 1–20; E. S. Coen and E. M. Meyerowitz, 1991, Nature 353: 31–37). However, HPDEF expression was also observed in ovule primordia and expression continued in developing ovules until anthesis, indicating that this gene may have a role in ovule development. Expression of HPDEF was not detected in megaspore mother cells, or in sexual or aposporous embryo sacs. In sexual Hieracium, HPDEF was uniformly expressed throughout the ovule integument until anthesis. In most ovules of the apomict, however, HPDEF expression was transiently down-regulated in a specific zone in the chalazal region where cells initiating aposporous embryo sac formation differentiate. Uniform low-level HPDEF expression was subsequently observed prior to anthesis in ovules from sexual and apomictic plants. HPDEF may be down-regulated as a consequence of apomictic initiation and/or its down-regulation may facilitate progression of apomictic events. Received: 15 September 1999 / Accepted: 12 October 1999     

Comparative cyto-embryological investigations of sexual and apomictic dandelions (Taraxacum) and their apomictic hybrids

. In the autonomous apomictic Taraxacum officinale (common dandelion), parthenogenetic egg cells develop into embryos and central cells into endosperm without prior fertilisation. Unreduced (2n) megaspores are formed via meiotic diplospory, a nonreductional type of meiosis. In this paper, we describe the normal developmental pathways of sexual and apomictic reproduction and compare these with the development observed in the apomictic hybrids. In sexual diploids, a standard type of megasporogenesis and embryo sac development is synchronised between florets in individual capitula. In contrast, we observed that megasporogenesis and gametogenesis proceeded asynchronously between florets within a single capitulum of natural triploid apomicts. In addition, autonomous endosperm and embryo development initiated independently within individual florets. Parthenogenetic initiation of embryo development in outdoor apomicts was found to be temperature-dependent. Egg cells produced in natural apomicts were not fertilised after pollination with haploid pollen grains although pollen tubes were observed to grow into their embryo sacs. Both reductional and diplosporous megasporogenesis were observed in individual inflorescences of triploid apomictic hybrids. Embryo and endosperm development initiated independently in natural and hybrid apomicts.     

Apomixis in hawkweed: Mendel's experimental nemesis

Mendel used hawkweeds and other plants to verify the laws of inheritance he discovered using Pisum. Trait segregation was not evident in hawkweeds because many form seeds asexually by apomixis. Meiosis does not occur during female gametophyte formation and the mitotically formed embryo sacs do not require fertilization for seed development. The resulting progeny retain a maternal genotype. Hawkweeds in Hieracium subgenus Pilosella form mitotic embryo sacs by apospory. The initiation of sexual reproduction is required to stimulate apospory in ovules and to promote the function of the dominant locus, LOSS OF APOMEIOSIS, which stimulates the differentiation of somatic aposporous initial (AI) cells near sexually programmed cells. As AI cells undergo nuclear mitosis the sexual pathway terminates. The function of the dominant locus LOSS OF PARTHENOGENESIS in aposporous embryo sacs enables fertilization-independent embryo and endosperm development. Deletion of either locus results in partial reversion to sexual reproduction, and loss of function in both loci results in reversion to sexual development. In these apomicts, sexual reproduction is therefore the default reproductive mode upon which apomixis is superimposed. These loci are unlikely to encode factors critical for sexual reproduction but might recruit the sexual pathway to enable apomixis. Incomplete functional penetrance of these dominant loci is likely to lead to the generation of rare sexual progeny also derived from these facultative apomicts.     

Heterochronic expression of sexual reproductive programs during apomictic development in Tripsacum

Some angiosperms reproduce by apomixis, a natural way of cloning through seeds. Apomictic plants bypass both meiosis and egg cell fertilization, producing progeny that are genetic replicas of the mother plant. In this report, we analyze reproductive development in Tripsacum dactyloides, an apomictic relative of maize, and in experimental apomictic hybrids between maize and Tripsacum. We show that apomictic reproduction is characterized by an alteration of developmental timing of both sporogenesis and early embryo development. The absence of female meiosis in apomictic Tripsacum results from an early termination of female meiosis. Similarly, parthenogenetic development of a maternal embryo in apomicts results from precocious induction of early embryogenesis events. We also show that male meiosis in apomicts is characterized by comparable asynchronous expression of developmental stages. Apomixis thus results in an array of possible phenotypes, including wild-type sexual development. Overall, our observations suggest that apomixis in Tripsacum is a heterochronic phenotype; i.e., it relies on a deregulation of the timing of reproductive events, rather than on the alteration of a specific component of the reproductive pathway.     

Sexual tetraploid and apomictic pentaploid populations ofHieracium pilosella (Compositae)

Five species are recognized inHieracium subgen.Pilosella sect.Pilosellina Fries. Four are diploid (2x, 2n = 18), one (H. pilosella L.) is highly variable morphologically and cytologically (from 2x to 10x), in its mode of reproduction (self-incompatibility, agamospermy, amphimixis, apo-amphimixis) and in its hybridization pattern. A part of this huge agamic complex was analysed by comparing sexual 4x and apomictic 5x plants (crossing and germination experiments, measurements of vegetative reproduction by stolons etc.). In the experimental garden apomictic 5x produced more stolons than the sexual 4x plants and the total length of the stolons per rosette was greater. However, in nature, the competitive potential of the sexual plants seems to be higher, presumably as a result of the higher mortality of ramets in 5x. Sexual 4x plants often grow in dense and grazed grass vegetation, whereas 5x apomicts often occur in dunes with patchy vegetation. Apomicts produce more capitula per rosette, and sexual rosettes form only about 60% of the number of viable achenes as compared to apomictic ones. Therefore, apomicts appear to be characterized by a greater colonizing ability than sexual plants. Apomictic plants produce equal numbers of viable achenes under conditions of both open pollination and isolation. Sexual plants do not form any viable achenes after isolation and produce a somewhat lower percentage of achenes after open pollination than do apomictics. 5xreproduce exclusively apomictically. Apo-amphimixis was never observed in pentaploids and only very rarely in tetraploids. Addition hybrids are very rare. The cross sexual 4x × apomictic 5x failed in 70% of the attempts, but the recombination of genomes carrying genes for apomixis is possible and results in apomictic 4x and sexual 5x, both with a reduced number of viable achenes. In nature sexual and apomictic plants may occur in close proximity. In such cases the germination rate of the achenes of 4x and 5x is lower; this may indicate that apomictic plants fertilize sexual plants in nature (unidirectional gene-flow). 5x plants form euploid gametes carrying two or three genomes. The results of the crossing experiments can be explained in terms ofNogler's theory of monogenic inheritance of apospory.Variation and evolution inHieracium subg.Pilosella sect.Pilosellina I.     

<Emphasis Type="Italic">Hieracium</Emphasis> subgen. <Emphasis Type="Italic">Pilosella</Emphasis>: pollen stainability in sexual,apomictic and sterile plants

Two pollen stainability tests (Alexander’s stain and acetocarmine) were used to detect differences in pollen viability of the sexual, apomictic and sterile plants of Hieracium subgen. Pilosella. In sexual taxa (Hieracium bauhini and H. densiflorum), the average stainability was 93.7–98.4%. Similarly high stainability (92.2–97.2%) was found in the apomictic Hieracium pilosellinum and in the majority of the apomictic populations (or plants) of the pentaploid and hexaploid H. bauhini. In some apomictic plants of Hieracium bauhini the average pollen stainability was 49.0–75.4%. The lowest pollen stainability was found in the sterile plants, i.e. the triploid H. pistoriense (33.6%) and the pentaploid H. brachiatum (29.6%).     

An <Emphasis Type="Italic">Hieracium</Emphasis> mutant, <Emphasis Type="Italic">loss of</Emphasis><Emphasis Type="Italic">apomeiosis 1</Emphasis> (<Emphasis Type="Italic">loa1</Emphasis>) is defective in the initiation of apomixis

Asexual seed formation (apomixis) in Hieracium aurantiacum occurs by mitotic embryo sac formation without prior meiosis in ovules (apomeiosis), followed by fertilization-independent embryo and endosperm development. Sexual reproduction begins first in Hieracium ovules with megaspore mother cell (MMC) formation. Apomixis initiates with the enlargement of somatic cells, termed aposporous initial (AI) cells, near sexual cells. AI cells grow towards sexually programmed cells undergoing meiosis, which degrade as the dividing nuclei of AIs obscure and displace them. Following Agrobacterium-mediated transformation of an aneuploid Hieracium aurantiacum apomict, a somaclonal mutant designated “loss of apomeiosis 1” (loa1) was recovered, which had significantly lost the ability to form apomictic seed. Maternal apomictic progeny were rare and low levels of germinable seedlings were primarily derived from meiotically derived eggs. Cytological analysis revealed defects in AI formation and function in loa1. Somatic cells enlarged some distance away from sexual cells and unlike AI cells, these expanded away from sexual cells without nuclear division. Surprisingly, many accumulated callose in the walls, a marker associated with meiotically specified cells. These defective AI (DAI) cells only had partial sexual identity as they failed to express a marker reflecting entry to meiosis that was easily detected in MMCs and they ultimately degraded. DAI cell formation did not lead to a compensatory increase in functional sexual embryo sacs, as collapse of meiotic embryo sacs was prevalent in the aneuploid somaclonal mutant. Positional cues that are important for AI cell differentiation, growth and fate may have been disrupted in the loa1 mutant and this is discussed. The authors Takashi Okada, Andrew S. Catanach and Susan D. Johnson made equal contributions to the data.     

Sporophytic ovule tissues modulate the initiation and progression of apomixis in Hieracium

Apomixis in Hieracium subgenus Pilosella initiates in ovules when sporophytic cells termed aposporous initial (AI) cells enlarge near sexual cells undergoing meiosis. AI cells displace the sexual structures and divide by mitosis to form unreduced embryo sac(s) without meiosis (apomeiosis) that initiate fertilization-independent embryo and endosperm development. In some Hieracium subgenus Pilosella species, these events are controlled by the dominant LOSS OF APOMEIOSIS (LOA) and LOSS OF PARTHENOGENESIS (LOP) loci. In H. praealtum and H. piloselloides, which both contain the same core LOA locus, the timing and frequency of AI cell formation is altered in derived mutants exhibiting abnormal funiculus growth and in transgenic plants expressing rolB which alters cellular sensitivity to auxin. The impact on apomictic and sexual reproduction was examined here when a chimeric RNAse gene was targeted to the funiculus and basal portions of the ovule, and also when polar auxin transport was inhibited during ovule development following N-1-naphthylphthalamic acid (NPA) application. Both treatments led to ovule deformity in the funiculus and distal parts of the ovule and LOA-dependent alterations in the timing, position, and frequency of AI cell formation. In the case of NPA treatment, this correlated with increased expression of DR5:GFP in the ovule, which marks the accumulation of the plant hormone auxin. Our results show that sporophytic information potentiated by funiculus growth and polar auxin transport influences ovule development, the initiation of apomixis, and the progression of embryo sac development in Hieracium. Signals associated with ovule pattern formation and auxin distribution or perception may influence the capacity of sporophytic ovule cells to respond to LOA.     

DOES HYBRIDIZATION DRIVE THE TRANSITION TO ASEXUALITY IN DIPLOID BOECHERA?

Gametophytic apomixis is a common form of asexual reproduction in plants. Virtually all gametophytic apomicts are polyploids, and some view polyploidy as a prerequisite for the transition to apomixis. However, any causal link between apomixis and polyploidy is complicated by the fact that most apomictic polyploids are allopolyploids, leading some to speculate that hybridization, rather than polyploidy, enables apomixis. Diploid apomixis presents a rare opportunity to isolate the role of hybridization, and a number of diploid apomicts have been documented in the genus Boechera (Brassicaceae). Here, we present the results of a microsatellite study of 1393 morphologically and geographically diverse diploid individuals, evaluating the hypothesis that diploid Boechera apomicts are hybrids. This genus‐wide dataset was made possible by the applicability of a core set of microsatellite loci in 69 of the 70 diploid Boechera species and by our ability to successfully genotype herbarium specimens of widely varying ages. With few exceptions, diploid apomicts exhibited markedly high levels of heterozygosity resulting from the combination of disparate genomes. This strongly suggests that most apomictic diploid Boechera lineages are of hybrid origin, and that the genomic consequences of hybridization allow for the transition to gametophytic apomixis in this genus.     

The best of both worlds: Apomixis and sexuality co-occur in species of Microlicia,Melastomataceae

Apomixis, the asexual formation of embryos and seeds, occurs in approximately 18% of angiosperm families. Melastomataceae exhibits a remarkable number of apomictic species, distributed among different tribes. This mode of reproduction has been elucidated in Miconieae, but remains unclarified for other groups, such as Microlicieae. Although apomixis has been previously described for Microlicieae species, the cytological basis for this phenomenon is entirely unknown in this group. Thus, populations of Microlicia fasciculata and M. polystemma were used in order to (a) investigate the presence of autonomous apomixis; (b) verify if this mode of reproduction leads to polyembryony; and (c) investigate whether apomixis may occur in parallel with the sexual process. We tested these species for autonomous fruit set and polyembryony, and pollen viability, and analyzed pollen tube growth. Anatomical techniques were used to elucidate the micro- and megasporogenesis and gametogenesis. The species showed autonomous fruit and seed formation and exhibited polyembryony. Apospory and adventitious embryony were the developmental mechanisms of apomixis in M. fasciculata and M. polystemma, respectively. Both species exhibited low pollen viability. However, some viable pollen, reduced embryo sac formation, natural pollination and pollen tube growth enable sexual reproduction and characterize these species as facultative apomicts. The independence of pollinators for fruit set, uniparental reproduction and the possibility of sexual reproduction, confer reproductive assurance and flexibility, bringing together advantages of sexual and asexual reproduction. In this sense, apomixis may have played an important role in the evolution and diversification of Microlicia, a widely distributed genus in the Brazilian Cerrado.     

Sexual Hieracium pilosella plants are better inter-specific,while apomictic plants are better intra-specific competitors

Apomixis, asexual reproduction through seeds, occurs in over 40 plant families. This widespread phenomenon can lead to the fixation of successful genotypes, resulting in a fitness advantage. On the other hand, apomicts are expected to lose their fitness advantage if the environment changes because of their limited evolutionary potential, which is due to low genetic variability and the potential accumulation of deleterious somatic mutations. Nonetheless, some apomicts have been extremely successful, for example certain apomictic accessions of Hieracium pilosella L. from New Zealand, where the plant is invasive. Here, we investigate whether the success of these apomictic accessions could be due to a fitness advantage by comparing the vegetative competitiveness of apomictic H. pilosella from New Zealand with sexual accessions of H. pilosella from Europe. Sexual and apomictic plants were grown either (A) alone (no competition), (B) in competition with the other type (intra-specific competition), (C) in competition with the grass Bromus erectus (inter-specific competition), and (D) in competition with the other type and the grass B. erectus (intra- and inter-specific competition). To distinguish effects of apomixis and the region of origin, different H. pilosella lineages were compared. Furthermore, experiments were carried out to investigate effects of the ploidy level. We show that sexual plants are better inter-specific competitors than apomicts in terms of vegetative reproduction (number of stolons) and vegetative spread (stolon length), while apomicts do better than sexuals in intra-specific competition. The magnitude of the effect was in some cases dependent on the ploidy levels of the plants. Furthermore, apomicts always produced more stolons than sexuals, suggesting potential displacement of sexuals by apomicts where they co-occur.