The occurrence of phenotypically complementary apomixis-recombinants in crosses between sexual and apomictic dandelions (<Emphasis Type="Italic">Taraxacum officinale</Emphasis>)

Apomictic seed development in dandelion ( Taraxacum officinale) involves (1) restitutional meiosis (diplospory), (2) egg cell parthenogenesis, and (3) autonomous endosperm development. The question is whether these elements of apomixis are controlled by one single gene or by several independent genes. Five triploid non-apomictic hybrids, obtained in diploid sexual × triploid apomict crosses were characterized using cyto-embryological and genetic methods. Nomarski-differential interference contrast microscopy and the transmission of microsatellite markers and ploidy levels indicated that the hybrids combined elements of the apomictic and the sexual developmental pathway. Hybrids form two complementary groups with respect to the presence or absence of parthenogenesis and autonomous endosperm development. The occurrence of complementary apomixis-recombinants suggests that parthenogenesis and autonomous endosperm development in Taraxacum are regulated independently by different genes. This study also indicates that early embryo development is independent of endosperm formation, but that endosperm is essential for later embryo growth.     

Sexual reproduction, hybridization, apomixis, and polyploidization in the genus Boechera (Brassicaceae)

Of the 340 genera in the Brassicaceae, apomictic reproduction is found only in the North American genus Boechera. We investigated phylogenetic relationships, ability to hybridize, mating system, and ploidy levels of 92 lines sampled from 85 populations and representing 19 Boechera species. Phylogenetic analyses based on chloroplast DNA sequences identified three lineages in the genus. Reciprocal crosses of each line were made to a common sexual diploid B. stricta tester. The resulting F(1) progeny were analyzed for the inheritance of polymorphic microsatellite loci, genome size, and seed production. Intraspecific B. stricta crosses confirmed that this species is mostly diploid and sexual. Interspecific crosses revealed many other species were diploid and sexual and could be successfully hybridized with the tester. We also found obligate and facultative apomictic diploid and triploid lines. De novo F(1) polyploids (either triploids or tetraploids) were derived from the union of nonreduced (from an apomictic parent) and reduced (from the tester) gametes. However, seed production of these F(1) plants was generally low, suggesting a failure in the transmission of apomixis. The creation of a wide array of segregating genetic populations will facilitate future research on the evolution and inheritance of quantitative variation in Boechera.     

Tripsacum dactyloides (Poaceae): a natural model system to study parthenogenesis

Diplosporous apomeiosis, formation of unreduced embryo sacs primarily of the Antennaria type, followed by parthenogenetic embryo development and pseudogamy (fertilization of the central cell) describe gametophytic apomixis within the Tripsacum agamic complex. Tripsacum dactyloides (Eastern gamagrass) is a close relative of domesticated maize and was chosen as a natural model system to investigate gene expression patterns associated with parthenogenesis. The genome size of diploid sexual and polyploid apomictic T. dactyloides was estimated by flow cytometry to be 7.37 pg (2C), 14.74 pg (4C) and 22.39 pg (6C), respectively. The diploid genome size is thus approximately 1.352 larger than that of maize. The apomeiotic-pseudogamous pathway of seed formation was demonstrated at a rate of 92% by the flow cytometric seed screen (FCSS) with single mature seeds in tetraploid accessions. This number includes twin embryos which were detected in 13% of the seeds analyzed. Fertilization of unreduced egg cells (B_III hybrids) was measured in 10% of apomictic seeds. Autonomous (fertilization-independent) embryo development and fertilization-dependent endosperm formation were confirmed by pollination of tetraploid T. dactyloides with a diploid transgenic maize line carrying an actin::#-glucuronidase (GUS) reporter construct. GUS expression was detected after pollination in the developing endosperm, but not in the embryo. In similar intraspecific crossing experiments with maize, GUS expression was detected in both the embryo and endosperm. A protocol was established for microdissection of embryo sacs and early parthenogenetic embryos of T. dactyloides. Together, these techniques provide new tools for future studies aimed at comparing gene expression patterns between sexual maize and sexual or apomictic T. dactyloides.     

Reconstruction of reproductive diversity in Hypericum perforatum L. opens novel strategies to manage apomixis

The mode of reproduction was characterized for 113 accessions of the tetraploid facultative apomictic species Hypericum perforatum using bulked or single mature seeds in the flow cytometric seed screen (FCSS). This screen discriminates several processes of sexual or asexual reproduction based on DNA contents of embryo and endosperm nuclei. Seed formation in H. perforatum proved to be highly polymorphic. Eleven different routes of reproduction were determined. For the first time, individual seeds were identified that originated from two embryo sacs: the endosperm from an aposporous and the embryo from the legitimate meiotic embryo sac. Moreover, diploid plants were discovered, which apparently reproduce by a hitherto unknown route of seed formation, that is chromosome doubling within aposporous initial cells followed by double fertilization. Although most plants were tetraploid and facultative sexual/apomictic, diploid obligate sexuals and tetraploid obligate apomicts could be selected. Additionally, genotypes were detected which at a high frequency produced embryos either from reduced parthenogenetic or unreduced fertilized egg cells. The endosperm developed most frequently after fertilization of the central cell in aposporous embryo sacs (pseudogamy) but in few cases also autonomously. The genetic control of apomixis appears to be complex in H. perforatum. Basic material was developed for breeding H. perforatum, and strategies are suggested for elucidation of inheritance as well as evolution of apomixis and for molecular approaches of apomixis engineering.     

Differential effects of polyploidy and diploidy on fitness of apomictic Boechera

The co-occurrence of apomixis (asexual reproduction) and polyploidy in plants has been the subject of debate in regard to the origin and evolution of asexuality. In recent years, polyploidy has been postulated as a maintenance and stabilization factor rather than as a source of apomixis origin. It is assumed polyploidy facilitates the compensation for mutation accumulation, and hence, the rare occurrence of diploid apomixis indirectly supports this finding. Nevertheless, diploid apomicts exist and are successful, especially in the genus Boechera. While comparing phenotypic traits, fitness-related traits and apomixis penetrance between both diploid and triploid apomicts in the genus Boechera, it was expected to find trait variance that can be attributed to ploidy. Surprisingly, little trait variation could be assigned to ploidy, but rather trait variations were mainly genotype-specific. Additionally, it is shown that paternal contribution is very important for trait success, even though all offspring are genetically identical to the mother plant. This harbors implications for the introduction of apomixis into crop plants, considering the effects of paternal contribution during asexual reproduction. Nevertheless, polyploidy is an efficient way to buffer deleterious mutations, but the flexibility of diploid apomicts of the genus Boechera for rare sexual events contributes to their success in nature.     

Selection on apomictic lineages of Taraxacum at establishment in a mixed sexual–apomictic population

A species’ mode of reproduction, sexual or asexual, will affect its ecology and evolution. In many species, asexuality is related to polyploidy. In Taraxacum, apomicts are triploid, and sexuals are diploid. To disentangle the effects of ploidy level and reproductive mode on life‐history traits, we compared established apomictic Taraxacum genotypes with newly synthesized apomictic genotypes, obtained from diploid–triploid crosses. Diploid–triploid crossing is probably the way that most apomictic lineages originate. New genotypes had on average a much lower seed set than established genotypes. Established genotypes differed on average from new genotypes, in particular under shaded conditions: the established genotypes had longer leaves and flowered later. The differences between new and established triploids resembled the differences that have been found between sexual diploids and established apomictic triploids. We conclude that ploidy differences alone are not directly responsible for observed differences between sexual diploid and apomictic triploid dandelions.     

Segregation for sexual seed production in Paspalum as directed by male gametes of apomictic triploid plants

BACKGROUND AND AIMS: Gametophytic apomixis is regularly associated with polyploidy. It has been hypothesized that apomixis is not present in diploid plants because of a pleiotropic lethal effect associated with monoploid gametes. Rare apomictic triploid plants for Paspalum notatum and P. simplex, which usually have sexual diploid and apomictic tetraploid races, were acquired. These triploids normally produce male gametes through meiosis with a range of chromosome numbers from monoploid (n = 10) to diploid (n = 20). The patterns of apomixis transmission in Paspalum were investigated in relation to the ploidy levels of gametes. METHODS: Intraspecific crosses were made between sexual diploid, triploid and tetraploid plants as female parents and apomictic triploid plants as male parents. Apomictic progeny were identified by using molecular markers completely linked to apomixis and the analysis of mature embryo sacs. The chromosome number of the male gamete was inferred from chromosome counts of each progeny. KEY RESULTS: The chromosome numbers of the progeny indicated that the chromosome input of male gametes depended on the chromosome number of the female gamete. The apomictic trait was not transmitted through monoploid gametes, at least when the progeny was diploid. Diploid or near-diploid gametes transmitted apomixis at very low rates. CONCLUSIONS: Since male monoploid gametes usually failed to form polyploid progenies, for example triploids after 4x x 3x crosses, it was not possible to determine whether apomixis could segregate in polyploid progenies by means of monoploid gametes.     

Cytotype stability, facultative apomixis and geographical parthenogenesis in Ranunculus kuepferi (Ranunculaceae)

Background and Aims

Asexual organisms are more widespread in previously glaciated areas than their sexual relatives (‘geographical parthenogenesis’). In plants, this pattern is probably dependent on reproductive isolation and stability of cytotypes within their respective distribution areas. Both partial apomixis and introgressive hybridization potentially destabilize the spatial separation of sexual and apomictic populations. The wide distribution of apomicts may be further enhanced by uniparental reproduction which is advantageous for colonization. These factors are studied in the alpine species Ranunculus kuepferi.

Methods

Geographical distribution, diversity and mode of reproduction of cytotypes were assessed using flow cytometry and flow cytometric seed screening on samples from 59 natural populations of Ranunculus kuepferi. Seed set of cytotypes was compared in the wild.

Key Results

Diploid sexuals are confined to the south-western parts of the Alps, while tetraploid apomicts dominate in previously glaciated and in geographically isolated areas despite a significantly lower fertility. Other cytotypes (3x, 5x and 6x) occur mainly in the sympatric zone, but without establishing populations. The tetraploids are predominantly apomictic, but also show a partial apomixis via an uncoupling of apomeiosis and parthenogenesis in the seed material. Both pseudogamy and autonomous endosperm formation are observed which may enhance uniparental reproduction.

Conclusions

Diploids occupy a glacial relic area and resist introgression of apomixis, probably because of a significantly higher seed set. Among the polyploids, only apomictic tetraploids form stable populations; the other cytotypes arising from partial apomixis fail to establish, probably because of minority cytotype disadvantages. Tetraploid apomicts colonize previously devastated and also distant areas via long-distance dispersal, confirming Baker''s law of an advantage of uniparental reproduction. It is concluded that stability of cytotypes and of modes of reproduction are important factors for establishing a pattern of geographical parthenogenesis.     

Asexual reproduction in a close relative of Arabidopsis: a genetic investigation of apomixis in Boechera (Brassicaceae)

Understanding apomixis (asexual reproduction through seeds) is of great interest to both plant breeders and evolutionary biologists. The genus Boechera is an excellent system for studying apomixis because of its close relationship to Arabidopsis, the occurrence of apomixis at the diploid level, and its potentially simple inheritance by transmission of a heterochromatic (Het) chromosome. Diploid sexual Boechera stricta and diploid apomictic Boechera divaricarpa (carrying a Het chromosome) were crossed. Flow cytometry, karyotype analysis, genomic in situ hybridization, pollen staining and seed-production measurements were used to analyse the parents and resulting F1, F2 and selected F3 and test-cross (TC) generations. The F1 plant was a low-fertility triploid that produced a swarm of aneuploid and polyploid F2 progeny. Two of the F2 plants were fertile near-tetraploids, and analysis of their F3 and TC progeny revealed that they were sexual and genomically stabilized. The apomictic phenotype was not transmitted by genetic crossing as a single dominant locus on the Het chromosome, suggesting a complex genetic control of apomixis that has implications for future genetic and evolutionary analyses in this group.     

Inheritance of apomeiosis (diplospory) in fleabanes (Erigeron, Asteraceae)

Unreduced egg formation (apomeiosis) in flowering plants is rare except when it is coupled with parthenogenesis to yield gametophytic apomixis via apospory or diplospory. Results from genetic mapping studies in diverse apomictic taxa suggest that apomeiosis and parthenogenesis are genetically linked, a finding that is compatible with the conventional rationale that apomeiosis is unlikely to evolve independently because of deleterious fitness consequences. An Erigeron annuus (apomictic) x E. strigosus (sexual) genetic mapping population, however, included a high proportion of plants that were highly apomeiotic (diplosporous) but nonapomictic; that is, they lacked autonomous seed production. To evaluate the function and inheritance of diplospory in Erigeron, a diplosporous triploid (2n=3x=27) seed parent was crossed with a sexual diploid (2n=2x=18) E. strigosus pollen parent to produce an F1 of 31 plants. Chromosome numbers and molecular markers (AFLPs) document the inheritance of the maternal genome through unreduced eggs resulting in recombinant but predominantly (77%) tetraploid F1s (2n=4x=36; 2n+n, B(III)). Quantitative evaluation shows continuous variation in the proportion of diplosporous (vs meiotic) ovules (41-89%) in tetraploid F1s despite the presumed equal genetic contribution from the diplosporous mother. These findings demonstrate the functional independence of diplospory and suggest that variation in the trait in F1s is likely due to segregating paternal modifiers. In addition, of six aneuploid (4x-1, 4x-2) F1s, three lack a subset of maternal AFLP markers. These plants likely arose from aberrant megagametogenesis resulting in the loss of maternal chromatin prior to fertilization.     

On the origin and evolution of apomixis in Boechera

The genetic mechanisms causing seed development by gametophytic apomixis in plants are predominantly unknown. As apomixis is consistently associated with hybridity and polyploidy, these confounding factors may either (a) be the underlying mechanism for the expression of apomixis, or (b) obscure the genetic factors which cause apomixis. To distinguish between these hypotheses, we analyzed the population genetic patterns of diploid and triploid apomictic lineages and their sexual progenitors in the genus Boechera (Brassicaceae). We find that while triploid apomixis is associated with hybridization, the majority of diploid apomictic lineages are likely the product of intra-specific crosses. We then show that these diploid apomicts are more likely to sire triploid apomictic lineages than conspecific sexuals. Combined with flow cytometric seed screen phenotyping for male and female components of apomixis, our analyses demonstrate that hybridization is an indirect correlate of apomixis in Boechera.     

Cytogeography and reproduction of the Paspalum simplex polyploid complex

 Paspalum simplex is a grass distributed throughout the phytogeographic Chaco region in South America from which sexual diploid and apomictic tetraploid races have been reported. We analysed native populations to determine their homogeneity of ploidy level, and the relationship between geographic distribution, ploidy levels, and reproductive systems. The ploidy level was established for 379 plants from 32 wild populations. Tetraploidy and apomixis constitute the most common combination for this species all over the Chaco region. Apomictic hexaploid plants were found associated with 4x populations. Diploids were confined to a small sector of the region. One sexual triploid plant arose from seed harvested in a pure 2x population, and one apomictic 3x plant was found in a mixed 2x-4x population. The results suggest that P. simplex is a core agamic complex characteristic of the Chaco region from which other apomictic polyploid species of the subgenus Anachyris could have evolved. Received July 24, 2002; accepted September 12, 2002 Published online: December 11, 2002     

Can obligate apomixis and more stable reproductive assurance explain the distributional successes of asexual triploids in Hieracium alpinum (Asteraceae)?

• Although reproductive assurance has been suggested to be one of the most important factors shaping the differential distributional patterns between sexuals and asexuals (geographic parthenogenesis), it has only rarely been studied in natural populations of vascular plants with autonomous apomixis. Moreover, there are almost no data concerning the putative relationship between the level of apomictic versus sexual plant reproduction on one hand, and reproductive assurance on the other.
• We assessed the level of sexual versus apomictic reproduction in diploid and triploid plants of Hieracium alpinum across its distributional range using flow cytometric analyses of seeds, and compared the level of potential and realized seed set, i.e. reproductive assurance, between the two cytotypes under field and greenhouse conditions.
• Flow cytometric screening of embryos and endosperms of more than 4,100 seeds showed that diploids produced solely diploid progeny sexually, while triploids produced triploid progeny by obligate apomixis. Potential fruit set was much the same in diploids and triploids from the field and the greenhouse experiment. While in the pollination‐limited environment in the greenhouse apomictic triploids had considerably higher realized fruit set than sexual diploids, there was no significant difference between cytotypes under natural conditions. In addition, sexuals varied to a significantly larger extent in realized fruit set than asexuals under both natural and greenhouse conditions.
• Our results indicate that triploid plants reproduce by obligate apomixis, assuring more stable and predictable fruit reproduction when compared to sexual diploids. This advantage could provide apomictic triploids with a superior colonisation ability, mirrored in a strong geographic parthenogenesis pattern observed in this species.

     

Apomixis via recombination of genome regions for apomeiosis (diplospory) and parthenogenesis in Erigeron (daisy fleabane, Asteraceae)

Apomixis in daisy fleabanes (Erigeron annuus and E. strigosus) is controlled by two genetically unlinked loci that regulate, independently, the formation of unreduced female gametophytes (apomeiosis, diplospory) and autonomous seed formation (parthenogenesis). In this work, fully apomictic F2s were regenerated by crossing F1s bearing, separately, these two functional regions. Two triploid (3x = 2n = 27) highly diplosporous F1s served as seed parents to an aneuploid (2x + 1 = 2n = 19) meiotic pollen donor bearing four AFLP markers linked to parthenogenetic seed formation but producing only abortive embryos and endosperm. Of 408 hybrids, 21 (5.1%) produced seed. Nine of these putative apomicts were tetraploids (4x), likely combining an unreduced egg from the diplosporous seed parent and a haploid gamete from the pollen parent (3x + x). The other 12 hybrid apomicts were pentaploid, interpreted as arising from the fusion of an unreduced diplosporous egg with an unreduced sperm cell (3x + 2x). Analysis indicated that all but three of the 21 synthetic apomicts recombined markers linked to diplospory and parthenogenesis. In addition, three additional hybrids combined markers linked to the two functional regions but produced only aborted embryos. The apomicts varied in percentage of diplosporous ovules (4.7–95.3% of all ovules produced) and in percentage of ovules that developed into seed (3.8–58.0%). These results support the hypothesis that apomeiosis and autonomous seed formation are genetically distinct, and that the traits can be separated and recombined to create hybrids exhibiting apomixis at near wildtype levels.     

Biogeographic distribution of polyploidy and B chromosomes in the apomictic Boechera holboellii complex

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploid individuals, whereas apomictic individuals are diploid or triploid and may additionally have B chromosomes. Using flow cytometry and karyotype analysis, we have shown that B chromosomes (a) occur in both diploid and triploid apomictic B. holboellii, (b) may occur in triploid B. drummondii, and (c) are dispensable for the plant. Both diploid and triploid karyotypes are found in multiple chloroplast haplotypes of both species, suggesting that triploid forms have originated multiple times during the evolution of this complex. B chromosome carriers are found in geographically and genetically distinct populations, but it is unknown whether the extra chromosomes are shared by common descent (single origin) or have originated via introgressive hybridization and repeated transitions from diploidy to triploidy. Diploid plants containing the Bs reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. Finally, our analyses of pollen size and viability suggest that irregular chromosome segregation in some triploid lineages may lead to the generation of diploid individuals which carry the B chromosomes.     

Mendelian segregation for two-factor apomixis in Erigeron annuus (Asteraceae)

The inheritance of asexual seed development (apomixis) in Erigeron annuus (Asteraceae) was evaluated in a triploid (2n=3x=27) population resulting from a cross between an apomictic tetraploid (2n=4x=36) pollen parent and a sexual diploid (2n=2x=18) seed parent. Diplospory (unreduced female gametophyte formation) and autonomous development (embryo and endosperm together) segregated independently in the population yielding four distinct phenotype classes: (1) apomictic plants combining diplospory and autonomous development, (2) diplosporous plants lacking autonomous development, (3) meiotic plants with autonomous (though abortive) development and (4) meiotic plants lacking autonomous development. Each class was represented by approximately one-quarter of the population (n=117), thus corresponding to a two-factor genetic model with no linkage (chi(2)=2.59, P=0.11). Observations demonstrate that autonomous embryo and endosperm development (jointly) may occur in either reduced or unreduced egg cells. The cosegregation of the traits is attributed to tight linkage or pleiotropy. The data are consistent with the hypothesis that autonomous development in E. annuus is regulated by a single fertilization factor, F, which initiates development of both the embryo and the endosperm in the absence of fertilization.     

Is the aneuploid chromosome in an apomictic Boechera holboellii a genuine B chromosome?

The Boechera holboellii complex comprises B. holboellii and B. drummondii, both of which can reproduce through sex or apomixis. Sexuality is associated with diploidy, whereas apomictic individuals can either be diploid, aneuploid or triploid. Aneuploid individuals are found in geographically and genetically distinct populations and contain a single extra chromosome. It is unknown whether the supernumerary chromosomes are shared by common descent (single origin) or have originated via introgressive hybridizations associated with the repeated transition from diploidy to triploidy. Diploid plants containing the extra chromosome(s) reproduce apomictically, suggesting that the supernumerary elements are associated with apomixis. In this study we compared flow cytometry data, chromosome morphology, and DNA sequences of sexual diploid and apomictic aneuploids in order to establish whether the extra chromosome fits the classical concept of a B chromosome. Karyotype analyses revealed that the supernumerary chromosome in the metaphase complement is heterochromatic and often smaller than the A chromosomes, and differs in length between apomictic plants from different populations. DNA sequence analyses furthermore demonstrated elevated levels of non-synonymous substitutions in one of the alleles, likely that on the aneuploid chromosome. Although the extra chromosome in apomictic Boechera does not go through normal reductional meiosis, in which it may get eliminated or accumulated by a B-chromosome-specific process, its variable size and heterochromatic nature does meet the remaining criteria for a genuine B chromosome in other species. Its prevalence and conserved genetic composition nonetheless implies that this chromosome, if truly a B, may be atypical with respect to its influence on its carriers.     

Introgression of apomixis into sexual species is inhibited by mentor effects and ploidy barriers in the Ranunculus auricomus complex

Background and Aims

Apomictic plants maintain functional pollen, and via pollination the genetic factors controlling apomixis can be potentially transferred to congeneric sexual populations. In contrast, the sexual individuals do not fertilize apomictic plants which produce seeds without fertilization of the egg cells. This unidirectional introgressive hybridization is expected finally to replace sexuality by apomixis and is thought to be a causal factor for the wide geographical distribution of apomictic complexes. Nevertheless, this process may be inhibited by induced selfing (mentor effects) of otherwise self-incompatible sexual individuals. Here whether mentor effects or actual cross-fertilization takes place between diploid sexual and polyploid apomictic cytotypes in the Ranunculus auricomus complex was tested via experimental crosses.

Methods

Diploid sexual mother plants were pollinated with tetra- and hexaploid apomictic pollen donators by hand, and the amount of well-developed seed compared with aborted seed was evaluated. The reproductive pathways were assessed in the well-developed seed via flow cytometric seed screen (FCSS).

Key Results

The majority of seed was aborted; the well-developed seeds have resulted from both mentor effects and cross-fertilization at very low frequencies (1·3 and 1·6 % of achenes, respectively). Pollination by 4x apomictic pollen plants results more frequently in cross-fertilization, whereas pollen from 6x plants more frequently induced mentor effects.

Conclusions

It is concluded that introgression of apomixis into sexual populations is limited by ploidy barriers in the R. auricomus complex, and to a minor extent by mentor effects. In mixed populations, sexuality cannot be replaced by apomixis because the higher fertility of sexual populations still compensates the low frequencies of potential introgression of apomixis.Key words: Apomixis, Ranunculus auricomus, evolution, geographical parthenogenesis, crossing experiments, flow cytometry