Multiple hybrid origins, genetic diversity and population genetic structure of two endemic Sorbus taxa on the Isle of Arran, Scotland

Understanding the processes that have given rise to polyploid hybrid taxa is central to our understanding of plant evolution. In this study, we use an array of genetic markers in a population analysis to elucidate the hybrid origins of the Arran whitebeams Sorbus arranensis and S. pseudofennica, two woody plant taxa endemic to the Isle of Arran, Scotland. It has been proposed that S. arranensis was derived by hybridization between S. aucuparia and S. rupicola, and that subsequent hybridization between S. arranensis and S. aucuparia gave rise to S. pseudofennica. Analyses of species-specific isozyme, nuclear intron and chloroplast DNA markers confirm the proposed origin of S. arranensis, and indicate that S. aucuparia was the female parent in the hybridization. Analysis of microsatellite markers suggests that there have been at least three origins of S. arranensis on Arran. Microsatellite markers also support the proposed hypothesis for the origin of S. pseudofennica, and indicate at least five hybrid origins of this taxon. In total, three multilocus genotypes of S. arranensis and eight of S. pseudofennica were detected on Arran and multilocus genotypic diversity levels Hg were 0.09 and 0.63, respectively. Genetic differentiation (thetaST) values based on multilocus genotypes are substantial (0.344 and 0.470 for S. arranensis and S. pseudofennica, respectively) implying limited seed flow among populations. These results indicate that the endemic Sorbus taxa on Arran are the products of multiple and ongoing evolutionary events. This information must be incorporated into management policies for their future conservation.     

The origin of intermediate species of the genus Sorbus

The genus Sorbus in Europe contains five diploid species, Sorbus aria, Sorbus aucuparia, Sorbus torminalis, Sorbus chamaemespilus and Sorbus domestica, classified into five different subgenera. The subgenus Aria (or the S. aria aggregate) contains apomictic triploid and tetraploid species. Within the genus there are, in addition, a number of species, morphologically intermediate between plants of the four main subgenera, which are considered to reproduce partly, or exclusively, by apomixis. These are believed to have originated by hybridisation between species in the S. aria aggregate and a species from another subgenus (either S. aucuparia, S. torminalis or S. chamaemespilus). We have used restriction fragment length polymorphism (RFLP) analysis on a total of 178 Sorbus accessions to test this model. The genome relationships of the different groups have been assessed, and the hybrid nature of the major intermediate groups is unequivocally demonstrated. Polyploid species in the S. aria aggregate show genetic variation, indicating the possibility of multiple origins and/or facultative apomictic breeding behaviour. A major finding, confirmed by microsatellite analysis, is that the 'intermediate' species S. intermedia is shown to have genomes from S aria, S aucuparia and S torminalis. Polymorphic mitochondrial DNA markers were used to determine the direction of the crosses that gave rise to new 'hybrid species'; in the majority of cases the pollen was provided by the parent from the S aria aggregate.     

Variation in Two Sorbus Species Endemic to the Isle of Arran, Scotland

A study of the morphology of plants from four species of Sorbus,S. arraensis, S. pseudofennica, S. aucuparia and S. rupicola,two of them endemic to North Arran, suggested that the originof the two endemic groups could have been by hybridization. Variation between individuals within the hybrid groups indicatesthat the two groups, probably of hybrid origin (S. arranensisand S. pseudofennica), could each consist of a set of geneticallyisolated clones reproducing by apomixis, with the possibilityin the case of S. pseudofennica of occasional genetic interchangeby sexual reproduction. The overlap of characters between the two hybrid groups suggeststhat the inflow of genetic material by hybridization and geneticmodification in these groups is continuing. Sorbus arranensis Hedl., Sorbus pseudofennica E. F. Warb., Sorbus aucuparia L., Sorbus rupicola (Syme) Hedl., interspecific hybridization, apomixis     

Parentage of endemic Sorbus L. (Rosaceae) species in the British Isles: evidence from plastid DNA

The parentage of polyploid Sorbus species in the British Isles was investigated using plastid DNA microsatellites. Four hundred and fifty-three samples from 30 taxa were screened using six microsatellite fragments, which gave 28 haplotypes. The haplotypes formed groups clearly related to the ancestral diploids Sorbus aria , Sorbus aucuparia , and Sorbus torminalis . Species in the Sorbus aria group all had Aria haplotypes (with the exception of one English S. aria ), species in the Sorbus anglica group had an Aucuparia haplotype, and species in the Sorbus latifolia group had a Torminalis haplotype. Sorbus intermedia had an Aucuparia haplotype. This indicated that the hybridization events that led to the formation of species in the S. anglica and S. latifolia groups usually did so with S. aria s.l . as the pollen-donating (paternal) parent. The polyploids S. anglica , Sorbus bristoliensis , Sorbus croceocarpa , Sorbus decipiens , Sorbus devoniensis , Sorbus hibernica , Sorbus lancastriensis , Sorbus leptophylla , Sorbus leyana , Sorbus minima , Sorbus rupicola , Sorbus subcuneata , Sorbus vexans , Sorbus whiteana , Sorbus wilmottiana , and three unnamed taxa may each be derived from a single maternal lineage. The polyploids Sorbus eminens , Sorbus porrigentiformis , and S. latifolia have multiple maternal lineages. The two primary diploid hybrids S . ×  thuringiaca and S . ×  vagensis have arisen many times independently.  © 2007 The Linnean Society of London, Botanical Journal of the Linnean Society , 2007, 154 , 291–304.     

Intertribal hybrid plants produced from crossing Arabidopsis thaliana with apomictic Boechera

Arabidopsis thaliana and Boechera belong to different tribes of the Brassicaceae and last shared a common ancestor 13-35 million years ago. A. thaliana reproduces sexually but some Boechera accessions reproduce by apomixis (asexual reproduction by seed). The two species are reproductively isolated, preventing introgression of the trait(s) controlling apomixis from Boechera into A. thaliana and their molecular characterisation. To identify if "escapers" from such hybridisation barriers exist, we crossed diploid or tetraploid A. thaliana mothers carrying a conditional male sterile mutation with a triploid Boechera apomict. These cross-pollinations generated zygotes and embryos. Most aborted or suffered multiple developmental defects at all stages of growth, but some seed matured and germinated. Seedlings grew slowly but eventually some developed into mature plants that were novel synthetic allopolyploid hybrids. With one exception, intertribal hybrids contained three Boechera plus either one or two A. thaliana genomes (depending on maternal ploidy) and were male and female sterile. The exception was a semi-fertile, sexual partial hybrid with one Boechera plus two A. thaliana genomes. The synthesis of "escapers" that survive rigorous early developmental challenges in crosses between A. thaliana and Boechera demonstrates that the inviability form of postzygotic reproductive isolation separating these distantly related species is not impenetrable. The recovery of a single semi-fertile partial hybrid also demonstrates that hybrid sterility, another form of postzygotic reproductive isolation, can be overcome between these species.     

Different contributions of birds and mammals to seed dispersal of a fleshy-fruited tree

Birds and mammals are important seed dispersers of fleshy-fruited plants. Although their behaviors are different, they frequently consume the same species. Thus to understand the dispersal of fleshy-fruited plants, the contribution of birds and mammals to seed dispersal has to be evaluated. Besides, within birds or mammals, some species may functionally different from others. In this study, we examined seed dispersal of the fleshy-fruited tree Swida controversa focusing on the difference between two frugivore groups (birds and mammals), and differences between species within groups. Collected seeds and S. controversa trees were identified using simple sequence repeat (SSR) genotyping, thus enabling to determine the distance between mother tree and dispersed seeds. The avian species were identified by DNA barcoding of feces, whereas the mammalian species were identified by the shape and smell of feces. Most seeds that fell near or under the maternal trees were dispersed by birds, resulting in short seed dispersal distances (average, 13 m). DNA barcoding detected five taxa of avian dispersers. No differences were detected in seed dispersal distance by different avian taxa (i.e., the distance between dispersed seeds and their maternal trees within the research plot); however the rate of seed immigration from outside the research plot by some avian taxa varied significantly. The seed dispersal distance by mammals was significantly further (127 m; min > 50 m) than that by birds. Additionally, immigrated seeds accounted for approximately two-thirds of mammal-dispersed seeds, indicating that these seeds were from outside the research plot, and that mammals significantly contributed to the long-distance seed dispersal of S. controversa. No differences in seed dispersal distance were detected between different mammalian taxa. Overall, this study revealed that birds and mammals show clearly different seed dispersal patterns, and thus, they play different roles in the regeneration of S. controversa.     

Biosystematic studies of Sorbus meinichii (Rosaceae) at Moster, S. Norway

Morphometric and cytological data were used to test the variation within the apomictic hybrid Sorbus meinichii from its type locality, in the Moster area, Bømlo in Sunnhordland, Norway. A lectotype is selected for the name. A total of 36 morphological characters were measured on leaves, stomata, flowers, pollen, fruits and seeds, from randomly selected S. meinichii individuals. In the field the material was classified into two groups, one including individuals of generally larger dimensions of leaves and fruits than the other. The preliminary classification was subsequently tested by a multivariate analysis of several characters, substantiating the existence of two morphs in the population at the type locality of S. meinichii , one corresponding to the selected lectotype. Assessment of the nature and taxonomic rank of these two morphs must await further studies. Cytological studies showed both forms of S. meinichii to be triploid, with 2n=51 chromosomes. The taxonomic separation of S. teodorii from S. meinichii on the basis of ploidy level is therefore not corroborated. Sorbus meinichii s. lat. was shown to be intermediate between its supposed parents, S. aucuparia and S. hybrida , in several leaf characters. All of the variation within the S. aucuparia × S. hybrida-complex in Fennoscandia is better included in one aggregate species Sorbus meinichii according to the present state of knowledge.     

Inheritance of the chloroplast genome in Sorbus aucuparia L. (Rosaceae)

Chloroplast DNA (cpDNA) inheritance was investigated in Sorbus aucuparia using progenies obtained from six controlled crosses between individuals of known haplotype. Polymerase chain reaction (PCR) amplification, followed by restriction analysis, was used to characterize 248 offspring for either of two polymorphic cpDNA fragments. All offspring exhibited the maternal haplotype, which indicates maternal inheritance of chloroplasts in S. aucuparia. Power analysis of the test showed that the frequency of paternal transmission of chloroplasts, if any, should not exceed 1.84% (with 99% confidence).     

ITS Polymorphisms Shed Light on Hybrid Evolution in Apomictic Plants: A Case Study on the Ranunculus auricomus Complex

The reconstruction of reticulate evolutionary histories in plants is still a major methodological challenge. Sequences of the ITS nrDNA are a popular marker to analyze hybrid relationships, but variation of this multicopy spacer region is affected by concerted evolution, high intraindividual polymorphism, and shifts in mode of reproduction. The relevance of changes in secondary structure is still under dispute. We aim to shed light on the extent of polymorphism within and between sexual species and their putative natural as well as synthetic hybrid derivatives in the Ranunculus auricomus complex to test morphology-based hypotheses of hybrid origin and parentage of taxa. We employed direct sequencing of ITS nrDNA from 68 individuals representing three sexuals, their synthetic hybrids and one sympatric natural apomict, as well as cloning of ITS copies in four representative individuals, RNA secondary structure analysis, and landmark geometric morphometric analysis on leaves. Phylogenetic network analyses indicate additivity of parental ITS variants in both synthetic and natural hybrids. The triploid synthetic hybrids are genetically much closer to their maternal progenitors, probably due to ploidy dosage effects, although exhibiting a paternal-like leaf morphology. The natural hybrids are genetically and morphologically closer to the putative paternal progenitor species. Secondary structures of ITS1-5.8S-ITS2 were rather conserved in all taxa. The observed similarities in ITS polymorphisms suggest that the natural apomict R. variabilis is an ancient hybrid of the diploid sexual species R. notabilis and the sexual species R. cassubicifolius. The additivity pattern shared by R. variabilis and the synthetic hybrids supports an evolutionary and biogeographical scenario that R. variabilis originated from ancient hybridization. Concerted evolution of ITS copies in R. variabilis is incomplete, probably due to a shift to asexual reproduction. Under the condition of comprehensive inter- and intraspecific sampling, ITS polymorphisms are powerful for elucidating reticulate evolutionary histories.     

SEED DEVELOPMENT IN CITRUS WITH SPECIAL REFERENCE TO 2X X 4X CROSSES

Comparative cytological and histological studies during embryogenesis of seeds from 2x X 2x and 2x x 4x crosses indicate that the ratio of ploidy level between embryo and endosperm is the most important factor affecting the course of seed development. The crosses produced seeds with the expected ploidy relationships between embryo, endosperm, and maternal tissue of 2:3:2 and 3:4:2 as well as the anomalous relationships 3:5:2, 4:6:2, and 6:10:2. All but 3:4:2 resulted in normal, germinable seeds. The ploidy level of the maternal tissue in relation to that of the embryo or endosperm did not appear to have any effect on seed development. About 92–99 % of seeds from 2x x 4x crosses containing triploid embryos with tetraploid endosperm aborted at different stages of embryogenesis. The abortion in all cases was preceded by abnormalities in the tetraploid endosperm. It is postulated that the unbalance of chromosome number between embryo and endosperm disturbs physiological relationships between these two tissues, leading first to the abortion of the endosperm and then of the embryo.     

The role of domestication and maternal effects on seed traits of crop–wild sunflower hybrids (Helianthus annuus)

Hybridisation between crops and their wild relatives may promote the evolution of weeds. Seed germination and dormancy are the earliest life‐history traits and are highly influenced by the maternal parent. However, the ecological role of the maternal effect on seed traits in the evolution of crop–wild hybrids has received little attention. In this study, we test the relative importance of maternal and hybridisation effects on seed traits of the first generation of crop–wild sunflower hybrids (Helianthus annuus). Seed germination was tested in two wild populations with contrasting dormancy, two cultivated materials and their reciprocal crosses at four different times after harvest and three different temperatures. Seed germination at each of the four times, after ripening response and secondary dormancy were recorded along with four morphological traits. Additionally, the pericarp anatomy was analysed with light and scanning electron microscopy. We observed strong maternal effects on all seed traits. Seed germination, morphology and pericarp anatomy differed largely between the crop and wild seeds and these traits in the crop–wild hybrids resembled their female parent. Slight but significant hybridisation effects were observed in germination, mainly in seeds produced on wild plants. Crop hybridisation changed seed germination, the after ripening response and secondary dormancy in the crop direction. Morphological and anatomical traits associated with domestication strongly correlated with the observed differences in seed germination and dormancy in crop–wild sunflower hybrids. The large maternal effects along with the evolutionary divergence in seed traits were responsible for the large phenotypic differences observed in crop–wild hybrids with the same genetic composition. Wild‐like seed traits of hybrids suggest that there are no barriers to crop gene introgression at the seed level whereas crop‐like seed traits could be strongly selected against, conditioning the selection of traits expressed later in the life cycle and in the next generations.     

Conservation of genetic diversity in British populations of the diploid endemic Coincya monensis ssp monensis (Isle of Man Cabbage): the risk of hybridisation with the tetraploid alien, Coincya monensis ssp cheiranthos

Coincya monensis is represented in the British flora by two, cytologically distinct subspecies. Coincya monensis ssp monensis is an endemic diploid with a coastal sand dune distribution that includes a number of isolated populations. Coincya monensis ssp cheiranthos is a tetraploid alien, well established in South Wales in early successional habitats. Both subspecies share similar life form traits, flowering times and pollinators. Cluster analysis and phylogenetic reconstruction based on sequences of the mitochondrial nad4 gene confirmed the distinction between alien and endemic taxa. Tetraploid populations carry more polymorphic RAPDs loci and their genetic diversity is partitioned more within than among populations. In contrast, C. monensis ssp monensis has a distinct population genetic structure. Analysis of the multilocus genetic data confirmed a structure of genetically isolated, endemic population clusters in Scotland, Arran, the Isle of Man and South Wales. Experimental hybridisation showed the two subspecies are interfertile. Multivariate analysis of RAPDs data resolved hybrids between alien and endemic clusters and hybrids contained a proportion of alien-specific polymorphic loci. Hybrids of alien maternal parentage contained the mitochondrial nad4 sequence characteristic of the alien subspecies. Since the alien subspecies can invade mobile sand dune communities from urban sites and compete for pollinators, there is a risk that alien and endemic populations will mix and introgress. Conservation of endemic genetic diversity in Britain will require protection for all C. monensis ssp monensis populations. Currently, the most disjunct endemic population in South Wales is most at risk from introgression.     

Genetic structure of three Sorbus latifolia (Lam.) Pers. taxa endemic to northern Bavaria

The Franconian Alb (Bavaria, Germany) is rich in endemic Sorbus taxa, considered as apomictic microspecies and derived by hybridization between Sorbus aria aggregate and Sorbus torminalis (Sorbus latifolia aggregate). Molecular studies using the AFLP technique, neighbour joining, Bayesian clustering, principal coordinate analysis (PCo) and voucher studies were used to investigate genetic structure and origin of adult plants and cultivated offspring of three endemic S. latifolia taxa, namely Sorbus cordigastensis, Sorbus franconica and Sorbus adeana and probable parental species from the S. aria agg. and S. torminalis. The S. latifolia taxa, adults and progeny, showed low genetic variability and a more or less clonal structure, confirming an apomictic mode of reproduction. The investigated S. latifolia taxa were remarkably different among each other, confirming their status as microspecies. The AFLP data confirmed the hybrid origin of the S. latifolia taxa, they were 1.3–1.5 times more closely related to S. aria agg. than to S. torminalis. The S. aria agg. showed a complicate genetic structure and fell into four main groups, two intermediate groups besides Sorbus pannonica and Sorbus aria sensu stricto (S. aria s.str.). Some progeny of S. pannonica was more variable than expected and clustered partly with other groups indicating gene flow within S. aria agg. Different subgroups of the S. aria aggregate may be parental for the S. latifolia taxa, contributing to the remarkable genetic distances between them.     

Endosperm formation in aposporous Crataegus (Rosaceae, Spiraeoideae, tribe Pyreae): parallels to Ranunculaceae and Poaceae

Apomixis in Crataegus is primarily aposporous and requires pollination. The embryo sac is of the Polygonum type. A combination of meiotically unreduced embryo sacs with apparently reduced pollen would violate the usual requirement for a 2 : 1 ratio of maternal to paternal contributions to the endosperm. We therefore investigated the origin of endosperm in seeds of sexual diploids and apomictic polyploids of the sister genera Crataegus and Mespilus. Flow-cytometric DNA measurements from embryo and endosperm in mature seeds were converted to ploidy levels using leaf-tissue information. The diploids had triploid endosperm. In c. 60% of seed from polyploids, one sperm apparently contributes to the endosperm, while 25% or more may involve two sperm. Additional results suggest that trinucleate central cells also occur. Fertilization of meiotically unreduced eggs is indicated. The ratio of maternal to paternal contributions to the endosperm in these apomictic Crataegus is not constrained to 2 : 1. They thus resemble some Sorbus (Pyreae) and very distantly related Ranunculus (Ranunculaceae). It is suggested that Paspalum (Poaceae) may have similarly flexible endosperm ploidy levels.     

A model of triploid endosperm evolution driven by parent-offspring conflict

The parental investment in angiosperms comprises the endosperm, a nutrient reserve that is used during seed development. The endosperm contains genes from both parents. The most common endosperm form is the 3n Polygonum -type with more maternal genetic influence than paternal, i.e. with two maternal nuclei and one paternal nucleus. The evolutionary original state is thought to be a diploid endosperm with equal influence of the parents. We focus on the evolution of the triploid endosperm and show that a gene for triploid endosperm would have an initial advantage in a population of diploid endosperm type plants, and increase to fixation. We assume that endosperm amount is controlled by endosperm genes. Then a gene causing triploid endosperm will increase the influence of the mother plant on parental investment. The production of endosperm with two copies of the maternal genes will modify the inheritance of endosperm amount and cause an increased production of seeds.     

Comparative and Evolutionary Analysis of the Cytochrome b Sequences in Cyprinids with Different Ploidy Levels Derived from Crosses

The mitochondrial cyt b genes in the allotetraploid and triploid crucian carp as well as triploid common carp were isolated and completely sequenced. Their DNA sequences were compared with those derived from the cyt b genes of the red crucian carp, Japanese crucian carp, and common carp with MEGA 1.0 software. Phylogenetic analysis revealed the sister relationships between allotetraploid and diploid red crucian carp, between the triploid crucian carp and diploid Japanese crucian carp, and between triploid common carp and diploid common carp. Our results indicated the cyt b genes in the allotetraploid, triploid crucian carp, and triploid common carp were maternally inherited. Through maternal inheritance, the cyt b gene in the F11 tetraploid displayed extremely high similarity to that in the female parent red crucian carp after 11 generations (from F1 to F11 hybrids). Since the establishment of the new tetraploid stocks has great significance in analyzing evolutionary theory of vertebrate and in improving aquaculture industry, analysis of the cyt b gene and the elucidation of the variation of the cyt b gene DNA in different cyprinids prove that cyt b is a useful genetic marker to monitor the variations in the progeny of the crosses.     

Rabbit bait-take from plastic bait stations

Hybridisation is a rare event in facultatively apomictic species. We report the recovery of two hybrids from reciprocal crosses between the facultatively apomictic species Hieracium praealtum and H. caespitosum. Both parents were tetraploid (2n=4x=36). H. caespitosum x H. praealtum (CR6) was a hexaploid (2n=6x=54) and an apomict. The increased ploidy number is evidence of a BIII hybrid origin, having arisen from the fusion of a reduced and an unreduced gamete. In contrast, the hybrid recovered from the reciprocal cross H. praealtum x H. caespitosum (RC4) was a tetraploid and therefore probably arose as a BII hybrid fi-em the fusion of two reduced gametes. Further evidence for this is the expression of sexuality in this plant. As apomixis in Hieracium is thought to be determined by a single dominant locus, a sexual plant is consistent with a model of inheritance where this represents the putative homozygous recessive phenotype. The formation of a sexual plant from the hybridisation of apomicts has potentially significant evolutionary implications. The formation of an interspecific BIII hybrid has not previously been recorded.     

The basis of natural and artificial postzygotic hybridization barriers in Arabidopsis species

The success or failure of interspecific crosses is vital to evolution and to agriculture, but much remains to be learned about the nature of hybridization barriers. Several mechanisms have been proposed to explain postzygotic barriers, including negative interactions between diverged sequences, global genome rearrangements, and widespread epigenetic reprogramming. Another explanation is imbalance of paternally and maternally imprinted genes in the endosperm. Interspecific crosses between diploid Arabidopsis thaliana as the seed parent and tetraploid Arabidopsis arenosa as the pollen parent produced seeds that aborted with the same paternal excess endosperm phenotype seen in crosses between diploid and hexaploid A. thaliana. Doubling maternal ploidy restored seed viability and normal endosperm morphology. However, substituting a hypomethylated tetraploid A. thaliana seed parent reestablished the hybridization barrier by causing seed abortion and a lethal paternal excess phenotype. We conclude from these findings that the dominant cause of seed abortion in the diploid A. thaliana x tetraploid A. arenosa cross is parental genomic imbalance. Our results also demonstrate that manipulation of DNA methylation can be sufficient to erect hybridization barriers, offering a potential mechanism for speciation and a means of controlling gene flow between species.     

Melting pot of biodiversity: first insights into the evolutionary patterns of the Colchic bramble flora (Rubus subgenus Rubus,Rosaceae)

The Caucasus is a biodiversity hotspot of global significance, containing a number of highly diverse and species‐rich plant taxa. The region is also thought to be an important evolutionary hotspot for Rubus subgenus Rubus (brambles). However, Caucasian brambles have only been poorly studied to date and our knowledge of their evolutionary mechanisms, systematics and taxonomic variability remains rudimentary. Therefore, the objectives of this study were to shed light on the evolution, diversity and reproduction modes of Rubus in one of the two Caucasian glacial refugia, Colchis. Flow cytometry measurements were used to estimate DNA ploidy, a flow cytometric seed screen was conducted to determine reproduction mode and Sanger sequencing of two non‐coding plastid regions was used to reveal phylogenetic patterns. The most common ploidy level was tetraploid, followed by diploid and (rarely) triploid. Intra‐individual variation in reproduction mode was low, as the morphoseries Glandulosi and Radula exhibited strict sexuality and other taxa were mostly apomictic. A few exceptions were observed that deserve further attention, e.g. sexuality induced hypothetically by haploid pollen or by environmental conditions, a high proportion of triploid embryos or polyspermy. Plastid haplotype variability revealed specific, ancient evolutionary patterns with limited involvement of extant diploid taxa and recent isolation from European brambles. We provide the first insight into the variability and evolution of Colchic brambles, which serves as a starting point for further systematic and evolutionary studies.     

Direct evidence of pseudogamy in apomictic Brachiaria brizantha (Poaceae)

Brachiaria brizantha is a forage grass that has several apomictic accessions. B. brizantha cv. Marandu is a natural tetraploid aposporous apomict widely cultivated in Brazil. Pseudogamy was detected in this species by observation that seed set is suppressed in plants that have had the stigmas excised from the flowers. The egg cell develops parthenogenetically in the apomictic plants, meaning that fertilisation is necessary for the formation of the endosperm. A thorough knowledge of all the events of seed formation in natural apomictic plants is essential for a complete understanding of this mode of reproduction. In this paper, we show direct evidence of pseudogamy in B. brizantha through the cytological analysis of polar nucleus fertilisation and the determination of triploid level of the endosperm tissue. The development of the male gametophyte gives rise to a reduced tri-celled pollen, the viability of which varies throughout the year, reaching 88% in the peak of the flowering period. Discharge of the male gamete takes place around 10 h after pollination and monospermy is the predominant system observed. Precocious embryony was also observed in these plants; embryos arise from egg cells. Endosperm development followed the free nucleus model and was associated with the presence of an embryo. Cellularisation and reserve uptake occurred 2 days after pollination (DAP) and mature endosperm was observed 8 DAP. The triploid level of the endosperm in the apomictic accession confirmed the 2:1 maternal:paternal ratio of genome contribution in the tissue.