Is self-incompatibility a reproductive barrier for hybridization in a sympatric species?

PREMISE: Barriers at different reproductive stages contribute to reproductive isolation. Self-incompatibility (SI) systems that prevent self-pollination could also act to control interspecific pollination and contribute to reproductive isolation, preventing hybridization. Here we evaluated whether SI contributes to reproductive isolation among four co-occurring Opuntia species that flower at similar times and may hybridize with each other. METHODS: We assessed whether Opuntia cantabrigiensis, O. robusta, O. streptacantha, and O. tomentosa, were self-compatible and formed hybrid seeds in five manipulation treatments to achieve self-pollination, intraspecific cross-pollination, open pollination (control), interspecific crosses or apomixis, then recorded flowering phenology and synchrony. RESULTS: All species flowered in the spring with a degree of synchrony, so that two pairs of species were predisposed to interspecific pollination (O. cantabrigiensis with O. robusta, O. streptacantha with O. tomentosa). All species had distinct reproductive systems: Opuntia cantabrigiensis is self-incompatible and did not produce hybrid seeds as an interspecific pollen recipient; O. robusta is a dioecious species, which formed a low proportion of hybrid seeds; O. streptacantha and O. tomentosa are self-compatible and produced hybrid seeds. CONCLUSIONS: Opuntia cantabrigiensis had a strong pollen-pistil barrier, likely due to its self-incompatibility. Opuntia robusta, the dioecious species, is an obligate outcrosser and probably partially lost its ability to prevent interspecific pollen germination. Given that the self-compatible species can set hybrid seeds, we conclude that pollen-pistil interaction and high flowering synchrony represent weak barriers; whether reproductive isolation occurs later in their life cycle (e.g., germination or seedling survival) needs to be determined.

RH17 restricts reproductive fate and represses autonomous seed coat development in sexual Arabidopsis.

Plant sexual and asexual reproduction through seeds (apomixis) is tightly controlled by complex gene regulatory programs, which are not yet fully understood. Recent findings suggest that RNA helicases are required for plant germline development. This resembles their crucial roles in animals, where they are involved in controlling gene activity and the maintenance of genome integrity. Here, we identified previously unknown roles of Arabidopsis RH17 during reproductive development. Interestingly, RH17 is involved in repression of reproductive fate and of elements of seed development in the absence of fertilization. In lines carrying a mutant rh17 allele, development of supernumerary reproductive cell lineages in the female flower tissues (ovules) was observed, occasionally leading to formation of two embryos per seed. Furthermore, seed coat, and putatively also endosperm development, frequently initiated autonomously. Such induction of several features phenocopying distinct elements of apomixis by a single mutation is unusual and suggests that RH17 acts in regulatory control of plant reproductive development. Furthermore, an in-depth understanding of its action might be of use for agricultural applications.

Greater reproductive assurance of asexual plant compared with sexual relative in a low-density sympatric population: Experimental evidence for pollen limitation.

High reproductive assurance is regarded as a key advantage of uniparentally reproducing organisms for establishing a new population. This demographic benefit should especially be relevant for plants with autonomous apomixis, that is those which produce seeds completely independently from mates and pollinators. Indeed, many autonomous apomicts occupy larger distributional ranges when compared to their sexual relatives, showing geographical parthenogenesis patterns. However, uniparental reproduction advantage has only rarely been quantified in natural populations and results provided a mixed support, partly because allopatric sexual and asexual populations were exposed to different environmental and pollination conditions causing considerable between-population variation in the level of reproductive assurance. Here, we compared the level and stability of reproductive assurance between sexual self-incompatible and asexual autonomously apomictic plants of Hieracium alpinum (Asteraceae) cultivated in a sympatric low-density population with two levels of spatial clumping of sexual plants. Overall, we found that the realized seed set (i.e. proportion of well-developed seeds per capitulum) of asexuals was ca. 3 times greater than that of sexuals (83% vs. 27%), whereas the variance of this trait expressed as coefficient of variation was ca. 4 times smaller in asexuals compared with sexuals (19% vs. 83%). Solitary sexual plants had more than 2 times lower realized seed set when compared to clumps composed of two spatially close (20-30 cm) sexual plants (13% vs. 34%). Our study provides experimental evidence for benefit of uniparental reproduction of asexuals in a sympatric situation when the availability of mates is limited. This, together with unpredictability of pollinator environment could provide autonomous apomicts with an ultimate demographic superiority during colonization reflected in geographical parthenogenesis observed in this species.

The spread of infectious asexuality through haploid pollen.

The mechanisms of initiation and transmission of apomixis (asexual reproduction through seeds) in natural plant populations are important for understanding the evolution of reproductive variation. Here, we used the phylogenetic diversity of the genus Boechera (Brassicaceae), together with natural diversity in pollen types produced by apomictic lines, to test whether hybridization triggers the transition to asexuality, and whether a 'triploid bridge' is required for the formation of polyploid apomicts. We performed crosses between diploid sexual recipient and diploid apomictic donor lines and tested whether the mating system (interspecific hybridization vs intraspecific outcrossing) or pollen type (haploid vs diploid) influenced the transmission of apomixis from diploid apomictic donors into sexual recipients. We used genetic markers and flow cytometric analyses of embryo and endosperm in seeds to infer the reproductive mode. Within a single generation, initiation of both diploid and polyploid apomixis in sexual Boechera can occur. Diploid apomixis is transmitted through haploid pollen (infectious asexuality) and polyploids can form through multiple pathways. The three functional elements of apomixis occasionally segregate. Variation in pollen ploidy and the segregation of apomixis elements drive reproductive diversity of hybrids and outcrosses and can be utilized for apomixis initiation in crop breeding programs.

Establishing the cell biology of apomictic reproduction in diploid Boechera stricta (Brassicaceae).

Background and aims: In the Brassicaceae family, apomictic development is characteristic of the genus Boechera. Hybridization, polyploidy and environmental adaptation that arose during the evolution of Boechera may serve as (epi)genetic regulators of apomictic initiation in this genus. Here we focus on Boechera stricta, a predominantly diploid species that reproduces sexually. However, apomictic development in this species has been reported in several studies, indicating non-obligate sexuality. Methods: A progressive investigation of flower development was conducted using three accessions to assess the reproductive system of B. stricta. We employed molecular and cyto-embryological identification using histochemistry, transmission electron microscopy and Nomarski and epifluorescence microscopy. Key Results: Data from internal transcribed spacer (ITS) and chloroplast haplotype sequencing, in addition to microsatellite variation, confirmed the B. stricta genotype for all lines. Embryological data indicated irregularities in sexual reproduction manifested by heterochronic ovule development, longevity of meiocyte and dyad stages, diverse callose accumulation during meiocyte-to-gametophyte development, and the formation of triads and tetrads in several patterns. The arabinogalactan-related sugar epitope recognized by JIM13 immunolocalized to one or more megaspores. Furthermore, pollen sterility and a high frequency of seed abortion appeared to accompany reproduction of the accession ES512, along with the initiation of parthenogenesis. Data from flow cytometric screening revealed both sexual and apomictic seed formation. Conclusion: These results imply that B. stricta is a species with an underlying ability to initiate apomixis, at least with respect to the lines examined here. The existence of apomixis in an otherwise diploid sexual B. stricta may provide the genomic building blocks for establishing highly penetrant apomictic diploids and hybrid relatives. Our findings demonstrate that apomixis per se is a variable trait upon which natural selection could act.

Intergenerational environmental effects: functional signals in offspring transcriptomes and metabolomes after parental jasmonic acid treatment in apomictic dandelion.

Parental environments can influence offspring traits. However, the magnitude of the impact of parental environments on offspring molecular phenotypes is poorly understood. Here, we test the direct effects and intergenerational effects of jasmonic acid (JA) treatment, which is involved in herbivory-induced defense signaling, on transcriptomes and metabolomes in apomictic common dandelion (Taraxacum officinale). In a full factorial crossed design with parental and offspring JA and control treatments, we performed leaf RNA-seq gene expression analysis, LC-MS metabolomics and total phenolics assays in offspring plants. Expression analysis, leveraged by a de novo assembled transcriptome, revealed an induced response to JA exposure that is consistent with known JA effects. The intergenerational effect of treatment was considerable: 307 of 858 detected JA-responsive transcripts were affected by parental JA treatment. In terms of the numbers of metabolites affected, the magnitude of the chemical response to parental JA exposure was c. 10% of the direct JA treatment response. Transcriptome and metabolome analyses both identified the phosphatidylinositol signaling pathway as a target of intergenerational JA effects. Our results highlight that parental environments can have substantial effects in offspring generations. Transcriptome and metabolome assays provide a basis for zooming in on the potential mechanisms of inherited JA effects.

GID1 expression is associated with ovule development of sexual and apomictic plants.

KEY MESSAGE: BbrizGID1 is expressed in the nucellus of apomictic Brachiaria brizantha, previous to aposporous initial differentiation. AtGID1a overexpression triggers differentiation of Arabidopsis thaliana MMC-like cells, suggesting its involvement in ovule development. GIBBERELLIN-INSENSITIVE DWARF1 (GID1) is a gibberellin receptor previously identified in plants and associated with reproductive development, including ovule formation. In this work, we characterized the Brachiaria brizantha GID1 gene (BbrizGID1). BbrizGID1 showed up to 92% similarity to GID1-like gibberellin receptors of other plants of the Poaceae family and around 58% to GID1-like gibberellin receptors of Arabidopsis thaliana. BbrizGID1 was more expressed in ovaries at megasporogenesis than in ovaries at megagametogenesis of both sexual and apomictic plants. In ovules, BbrizGID1 transcripts were detected in the megaspore mother cell (MMC) of sexual and apomictic B. brizantha. Only in the apomictic plants, expression was also observed in the surrounding nucellar cells, a region in which aposporous initial cells differentiate to form the aposporic embryo sac. AtGID1a ectopic expression in Arabidopsis determines the formation of MMC-like cells in the nucellus, close to the MMC, that did not own MMC identity. Our results suggest that GID1 might be involved in the proper differentiation of a single MMC during ovule development and provide valuable information on the role of GID1 in sexual and apomictic reproduction.

Sexy males and sexless females: the origin of triploid apomicts.

Apomixis and polyploidy are closely associated in angiosperms, but the evolutionary reason for this association is unknown. Taraxacum officinale, the common dandelion, exists both as diploid sexuals and triploid apomicts. Here, in the context of T. officinale, we provide a model of the evolution of triploid apomicts from diploid sexuals. We posit an apomictic allele that arrests female meiosis in diploids, so that the plant produces diploid egg cells that can develop without fertilization, but haploid pollen. We propose occasional fertilization of diploid egg cells by haploid pollen, resulting in triploid apomicts that produce triploid egg cells but largely nonfunctional pollen. The irreversibility of this process renders diploid partial apomicts evolutionarily short-lived, and results in fixation of triploid apomicts except when they suffer extreme selective disadvantages. Our model can account for the high genetic diversity found in T. officinale triploid populations, because recombinant haploid pollen produced by diploids allows the apomictic allele to spread onto many genetic backgrounds. This leads to multiple clonal lineages in the newly apomictic population, and thereby alleviates some of the usual pitfalls of asexual reproduction.

Immunodetection of some pectic, arabinogalactan proteins and hemicellulose epitopes in the micropylar transmitting tissue of apomictic dandelions (Taraxacum, Asteraceae, Lactuceae).

In apomictic Taraxacum species, the development of both the embryo and the endosperm does not require double fertilisation. However, a structural reduction of ovular transmitting tissue was not observed in apomictic dandelions. The aim of this study was to analyse the chemical composition of the cell walls to describe the presence of arabinogalactan proteins (AGPs), hemicellulose and some pectic epitopes in the micropylar transmitting tissue of apomictic Taraxacum. The results point to (1) the similar distribution of AGPs in different developmental stages, (2) the absence of highly methyl-esterified homogalacturonan (HG) in transmitting tissue of ovule containing a mature embryo sac and the appearance of this pectin domain in the young seed containing the embryo and endosperm, (3) the similar pattern of low methyl-esterified pectin occurrence in both an ovule and a young seed with an embryo and endosperm in apomictic Taraxacum and (4) the presence of hemicelluloses recognised by LM25 and LM21 antibodies in the reproductive structure of Taraxacum.

Apomixis does not affect visitation to flowers of Melastomataceae, but pollen sterility does.

Apomixis is an asexual seed reproduction mechanism thorough which embryos are originated from material tissues inside the ovules, without precedent fertilisation. It allows plants to colonise new habitats, even in places where flower visitors are scarce or where plants are isolate. Apomixis seems to be related to pollen sterility and, in species with flowers that offer pollen as a reward for pollinators, the amount or quality of the pollen offered by these species may influence the amount of the visits and specific composition of the visitors. In order to test this hypothesis, we studied breeding systems of 16 species of Melastomataceae and their flower visitors, evaluating composition and abundance of the visits to apomictic and sexual species. Apomictic plants with no viable pollen or with pollen with low viability did not receive visits from pollinators, and consequently probably produce strictly apomictic fruits. On the other hand, apomictic and sexual plants with high pollen viability do receive visits; in this case, apomictic plants may produce fruits and seeds through both sexual and apomictic methods. The species composition of insects visiting Melastomataceae with high pollen viability was similar, regardless of whether the plants were apomictic or not. It seems that pollen viability levels are important to determine visits to the flowers irrespective of breeding system.

When sexual meets apomict: genome size, ploidy level and reproductive mode variation of Sorbus aria s.l. and S. austriaca (Rosaceae) in Bosnia and Herzegovina.

BACKGROUND AND AIMS: Allopolyploidy and intraspecific heteroploid crosses are associated, in certain groups, with changes in the mating system. The genus Sorbus represents an appropriate model to study the relationships between ploidy and reproductive mode variations. Diploid S. aria and tetraploid apomictic S. austriaca were screened for ploidy and mating system variations within pure and sympatric populations in order to gain insights into their putative causalities. METHODS: Flow cytometry was used to assess genome size and ploidy level among 380 S. aria s.l. and S. austriaca individuals from Bosnia and Herzegovina, with 303 single-seed flow cytometric seed screenings being performed to identify their mating system. Pollen viability and seed set were also determined. KEY RESULTS: Flow cytometry confirmed the presence of di-, tri- and tetraploid cytotype mixtures in mixed-ploidy populations of S. aria and S. austriaca. No ploidy variation was detected in single-species populations. Diploid S. aria mother plants always produced sexually originated seeds, whereas tetraploid S. austriaca as well as triploid S. aria were obligate apomicts. Tetraploid S. aria preserved sexuality in a low portion of plants. A tendency towards a balanced 2m : 1p parental genome contribution to the endosperm was shared by diploids and tetraploids, regardless of their sexual or asexual origin. In contrast, most triploids apparently tolerated endosperm imbalance. CONCLUSIONS: Coexistence of apomictic tetraploids and sexual diploids drives the production of novel polyploid cytotypes with predominantly apomictic reproductive modes. The data suggest that processes governing cytotype diversity and mating system variation in Sorbus from Bosnia and Herzegovina are probably parallel to those in other diversity hotspots of this genus. The results represent a solid contribution to knowledge of the reproduction of Sorbus and will inform future investigations of the molecular and genetic mechanisms involved in triggering and regulating cytotype diversity and alteration of reproductive modes.

De novo sequencing of the Hypericum perforatum L. flower transcriptome to identify potential genes that are related to plant reproduction sensu lato.

BACKGROUND: St. John's wort (Hypericum perforatum L.) is a medicinal plant that produces important metabolites with antidepressant and anticancer activities. Recently gained biological information has shown that this species is also an attractive model system for the study of a naturally occurring form of asexual reproduction called apomixis, which allows cloning plants through seeds. In aposporic gametogenesis, one or multiple somatic cells belonging to the ovule nucellus change their fate by dividing mitotically and developing functionally unreduced embryo sacs by mimicking sexual gametogenesis. Although the introduction of apomixis into agronomically important crops could have revolutionary implications for plant breeding, the genetic control of this mechanism of seed formation is still not well understood for most of the model species investigated so far. We used Roche 454 technology to sequence the entire H. perforatum flower transcriptome of whole flower buds and single flower verticils collected from obligately sexual and unrelated highly or facultatively apomictic genotypes, which enabled us to identify RNAs that are likely exclusive to flower organs (i.e., sepals, petals, stamens and carpels) or reproductive strategies (i.e., sexual vs. apomictic). RESULTS: Here we sequenced and annotated the flower transcriptome of H. perforatum with particular reference to reproductive organs and processes. In particular, in our study we characterized approximately 37,000 transcripts found expressed in male and/or female reproductive organs, including tissues or cells of sexual and apomictic flower buds. Ontological annotation was applied to identify major biological processes and molecular functions involved in flower development and plant reproduction. Starting from this dataset, we were able to recover and annotate a large number of transcripts related to meiosis, gametophyte/gamete formation, and embryogenesis, as well as genes that are exclusively or preferentially expressed in sexual or apomictic libraries. Real-Time RT-qPCR assays on pistils and anthers collected at different developmental stages from accessions showing alternative modes of reproduction were used to identify potential genes that are related to plant reproduction sensu lato in H. perforatum. CONCLUSIONS: Our approach of sequencing flowers from two fully obligate sexual genotypes and two unrelated highly apomictic genotypes, in addition to different flower parts dissected from a facultatively apomictic accession, enabled us to analyze the complexity of the flower transcriptome according to its main reproductive organs as well as for alternative reproductive behaviors. Both annotation and expression data provided original results supporting the hypothesis that apomixis in H. perforatum relies upon spatial or temporal mis-expression of genes acting during female sexual reproduction. The present analyses aim to pave the way toward a better understanding of the molecular basis of flower development and plant reproduction, by identifying genes or RNAs that may differentiate or regulate the sexual and apomictic reproductive pathways in H. perforatum.

Effects of apomixis and polyploidy on diversification and geographic distribution in Amelanchier (Rosaceae).

UNLABELLED: • PREMISE OF THE STUDY: Amelanchier polyploid apomicts differ from sexual diploids in their more complex diversification, greater species problems, and geographic distribution. To understand these differences, we investigated the occurrence of polyploidy and frequency of apomixis. This research helps clarify species delimitation in an evolutionarily complex genus.• METHODS: We used flow cytometry to estimate genome size of 1355 plants. We estimated the frequency of apomixis from flow-cytometrically determined ploidy levels of embryo and endosperm and from a progeny study using RAPD markers. We explored relationships of triploids to other ploidy levels and of ploidy levels to latitude plus elevation.• KEY RESULTS: Diploids (32% of sample) and tetraploids (62%) were widespread. Triploids (6%) mostly occurred in small numbers with diploids from two or more species or with diploids and tetraploids. Seeds from diploids were 2% apomictic, the first report of apomixis in Amelanchier diploids. Seeds from triploids were 75% apomictic. We documented potential triploid bridge and triploid block from unbalanced endosperm and low pollen viability. Seeds from tetraploids were 97% apomictic, and tetraploids often formed microspecies. We did not find strong evidence for geographical parthenogenesis in North American Amelanchier. Most currently recognized species contained multiple ploidy levels that were morphologically semicryptic.• CONCLUSIONS: Documentation of numerous transitions from diploidy to polyploidy helps clarify diversification, geographic distribution, and the species problem in Amelanchier. Despite the infrequent occurrence of triploids, their retention of 25% sexuality and capacity for triploid bridge may be important steps between sexual diploids and predominantly apomictic tetraploids.

Biogeographic variation in genetic variability, apomixis expression and ploidy of St. John's wort (Hypericum perforatum) across its native and introduced range.

BACKGROUND AND AIMS: St. John's wort (Hypericum perforatum) is becoming an important model plant system for investigations into ecology, reproductive biology and pharmacology. This study investigates biogeographic variation for population genetic structure and reproduction in its ancestral (European) and introduced (North America) ranges. METHODS: Over 2000 individuals from 43 localities were analysed for ploidy, microsatellite variation (19 loci) and reproduction (flow cytometric seed screen). Most individuals were tetraploid (93%), while lower frequencies of hexaploid (6%), diploid (<1%) and triploid (<1%) individuals were also identified. KEY RESULTS: A flow cytometric analysis of 24 single seeds per individual, and five individuals per population demonstrated opposite patterns between ploidy types, with tetraploids producing more apomictic (73%) than sexual (24%) seed, while hexaploids produced more sexual (73%) than apomictic (23%) seed. As hexaploids are derived from tetraploids, these data imply that gene dosage, in addition to the effects of hybridization, influences the switch from apomictic to sexual reproduction. No significant differences in seed production were found between Europe and North America. An analysis of population structure based upon microsatellite profiling demonstrated three major genetic clusters in Europe, whose distribution was reflective of Pleistocene glaciation (e.g. refugia) and post-glacial recolonization of Europe. CONCLUSIONS: The presence of pure and mixed populations representing all three genetic clusters in North America demonstrates that H. perforatum was introduced multiple times onto the continent, followed by gene flow between the different gene pools. Taken together, the data presented here suggest that plasticity in reproduction has no influence on the invasive potential of H. perforatum.

Pollen and stomata morphometrics and polyploidy in Eriotheca (Malvaceae-Bombacoideae).

Approximately 70% of the angiosperm species are polyploid, an important phenomenon in the evolution of those plants. But ploidy estimates have often been hindered because of the small size and large number of chromosomes in many tropical groups. Since polyploidy affects cell size, morphometric analyses of pollen grains and stomata have been used to infer ploidy level. Polyploidy is present in many species of the Cerrado, the Neotropical savanna region in Central Brazil, and has been linked to apomixis in some taxa. Eriotheca gracilipes and Eriotheca pubescens are common tree species in this region, and present cytotypes that form reproductive mosaics. Hexaploid individuals (2n = 6x = 276) are polyembryonic and apomictic, while tetraploid and diploid individuals (2n = 2x = 92, 2n = 4x = 184) are sexual and monoembryonic. We tested whether morphometric analysis can be used to estimate ploidy levels in E. gracilipes and E. pubescens individuals. Pollen material from diploid and hexaploid individuals of E. gracilipes, and tetraploid and hexaploid individuals of E. pubescens, were fixed in 50% FAA, and expanded leaves were dried in silica gel. Pollen grains and stomata of at least five individuals from each population were measured. The results demonstrate that all measures were significantly different among cytotypes. Individuals with higher levels of ploidy (hexaploid) all presented measurements that were higher than those with lower levels (diploid and tetraploid). There was no overlap between ploidy levels in each species at 95% confidence interval. Thus, the size of the pollen grains and stomata are effective parameters for analysis of ploidy levels in E. gracilipes and E. pubescens.

[Chromosomal structure of the hybrids between Allium cepa L. and Allium fistulosum L. with relative resistance to downy mildew based on in situ hybridization].

Genomic in situ hybridization (GISH) was used for a chromosomal composition study of the later generations of interspecific hybrids between A. cepa L. and A. fistulosum L., which are relatively resistant to downy mildew (peronosporosis). GISH revealed that F2 hybrids, which did not produce seeds, were triploids (2n = 3x = 24) with 24 chromosomes and possessed in their compliments 16 chromosomes of A. fistulosum L. and eight chromosomes of A. cepa L. or eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L. The advanced F5 hybrid, which produced few seeds, was amphidiploid with 32 chromosomes. BC1F5 hybrid was triploid with eight chromosomes of A. fistulosum L. and 16 chromosomes of A. cepa L., which did not produce seeds. BC2 (BC1F5) plant was amphidiploid that possessed 4 recombinant chromosomes and produced few seeds. GISH results point to 2n-gametes formation in macro- and microsporogenesis of the hybrids. The mechanism of 2n-gametes formation and the possibility of apomixes events in the backcrossing progeny are discussed.

The conserved chimeric transcript UPGRADE2 is associated with unreduced pollen formation and is exclusively found in apomictic Boechera species.

In apomictic Boechera spp., meiotic diplospory leads to the circumvention of meiosis and the suppression of recombination to produce unreduced male and female gametes (i.e. apomeiosis). Here, we have established an early flower developmental staging system and have performed microarray-based comparative gene expression analyses of the pollen mother cell stage in seven diploid sexual and seven diploid apomictic genotypes to identify candidate factors for unreduced pollen formation. We identified a transcript unique to apomictic Boechera spp. called UPGRADE2 (BspUPG2), which is highly up-regulated in their pollen mother cells. BspUPG2 is highly conserved among apomictic Boechera spp. genotypes but has no homolog in sexual Boechera spp. or in any other taxa. BspUPG2 undergoes posttranscriptional processing but lacks a prominent open reading frame. Together with the potential of stably forming microRNA-like secondary structures, we hypothesize that BspUPG2 functions as a long regulatory noncoding messenger RNA-like RNA. BspUPG2 has apparently arisen through a three-step process initiated by ancestral gene duplication of the original BspUPG1 locus, followed by sequential insertions of segmentally duplicated gene fragments, with final exonization of its sequence structure. Its genesis reflects the hybridization history that characterizes the genus Boechera.

Reproductive differentiation into sexual and apomictic polyploid cytotypes in Potentilla puberula (Potentilleae, Rosaceae).

BACKGROUND AND AIMS: Intraspecific reproductive differentiation into sexual and apomictic cytotypes of differing ploidy is a common phenomenon. However, mechanisms enabling the maintenance of both reproductive modes and integrity of cytotypes in sympatry are as yet poorly understood. This study examined the association of sexual and apomictic seed formation with ploidy as well as gene flow towards sexuals within populations of purely polyploid Potentilla puberula. METHODS: The study is based on 22 populations representing various combinations of five polyploid cytotypes (tetraploid-octoploid) from East Tyrol, Austria. Embryo ploidy and the endosperm/embryo ploidy ratio obtained by a flow cytometric seed screen were used to infer reproductive modes of seed formation and to calculate the male and female genomic contributions to the embryo and endosperm. Self-incompatibility (SI) patterns were assessed and a new indirect approach was used to test for the occurrence of intercytotype matings based on the variation in the male genomic contribution to sexually derived embryos on the level of developed seed. KEY RESULTS: Tetraploids formed seeds almost exclusively via sexual reproduction, whereas penta- to octoploids were preferentially apomictic. Non-random distribution of reproductive modes within maternal plants further revealed a tendency to separate the sexual from the apomictic mode among individuals. Self-incompatibility of sexuals indicated functionality of the gametophytic SI system despite tetraploidy of the nuclear genome. We found no indication for significant cross-fertilization of tetraploids by the high polyploids. CONCLUSIONS: The study revealed a rare example of intraspecific differentiation into sexual and apomictic cytotypes at the polyploid level. The integrity of the sexual tetraploids was maintained due to reproductive isolation from the apomictic higher polyploids. Functionality of the gametophytic SI system suggested that the tetraploids are functional diploids.

Recombination within the apospory specific genomic region leads to the uncoupling of apomixis components in Cenchrus ciliaris.

Apomixis enables the clonal propagation of maternal genotypes through seed. If apomixis could be harnessed via genetic engineering or introgression, it would have a major economic impact for agricultural crops. In the grass species Pennisetum squamulatum and Cenchrus ciliaris (syn. P. ciliare), apomixis is controlled by a single dominant "locus", the apospory-specific genomic region (ASGR). For P. squamulatum, 18 published sequenced characterized amplified region (SCAR) markers have been identified which always co-segregate with apospory. Six of these markers are conserved SCARs in the closely related species, C. ciliaris and co-segregate with the trait. A screen of progeny from a cross of sexual × apomictic C. ciliaris genotypes identified a plant, A8, retaining two of the six ASGR-linked SCAR markers. Additional and newly identified ASGR-linked markers were generated to help identify the extent of recombination within the ASGR. Based on analysis of missing markers, the A8 recombinant plant has lost a significant portion of the ASGR but continues to form aposporous embryo sacs. Seedlings produced from aposporous embryo sacs are 6× in ploidy level and hence the A8 recombinant does not express parthenogenesis. The recombinant A8 plant represents a step forward in reducing the complexity of the ASGR locus to determine the factor(s) required for aposporous embryo sac formation and documents the separation of expression of the two components of apomixis in C. ciliaris.

Male fertility versus sterility, cytotype, and DNA quantitative variation in seed production in diploid and tetraploid sea lavenders (Limonium sp., Plumbaginaceae) reveal diversity in reproduction modes.

The genus Limonium Miller, a complex taxonomic group, comprises annuals and perennials that can produce sexual and/or asexual seeds (apomixis). In this study, we used diverse cytogenetic and cytometric approaches to analyze male sporogenesis and gametogenesis for characterizing male reproductive output on seed production in Limonium ovalifolium and Limonium multiflorum. We showed here that the first species is mostly composed of diploid cytotypes with 2n = 16 chromosomes and the latter species by tetraploid cytotypes with 2n = 32, 34, 35, 36 chromosomes and had a genome roughly twice as big as the former one. In both species, euploid and aneuploid cytotypes with large metacentric chromosomes having decondensed interstitial sites were found within and among populations, possibly involved in chromosomal reconstructions. L. ovalifolium diploids showed regular meiosis resulting in normal tetrads, while diverse chromosome pairing and segregation irregularities leading to the formation of abnormal meiotic products are found in balanced and non-balanced L. multiflorum tetraploids. Before anther dehiscence, the characteristic unicellular, bicellular, or tricellular pollen grains showing the typical Limonium micro- or macro-reticulate exine ornamentation patterns were observed in L. ovalifolium using scanning electron microscopy. Most of these grains were viable and able to produce pollen tubes in vitro. In both balanced and unbalanced L. multiflorum tetraploids, microspores only developed until the "ring-vacuolate stage" with a collapsed morphology without the typical exine patterns, pointing to a sporophytic defect. These microspores were unviable and therefore never germinated in vitro. L. ovalifolium individuals presented larger pollen grains than those of L. multiflorum, indicating that pollen size and ploidy levels are not correlated in the Limonium system. Cytohistological studies in mature seeds from both species revealed that an embryo and a residual endosperm were present in each seed. Flow cytometric seed screens using such mature seeds showed quantitative variations in seeds ploidy level. It is concluded that male function seems to play an important role in the reproduction modes of Limonium diploids and tetraploids.