Interspecific Pollination Research Articles

Allogamy, metaxenia and hybrids in orchids, what do we know about it?

Orchids are a diverse group of plants, also manifested in their great diversity of flowers. Despite this, orchids are pollinated either through autogamy or allogamy (geitonogamy and xenogamy). Although there are some autogamous orchids, the majority are allogamous, mainly pollinated by xenogamy since they present physical or genetic barriers that prevent self-pollination. In addition, orchids are known for their capacity for interspecific pollination, which could influence fruits (metaxenia) and seeds (xenia) production. Its capacity for hybridization represents an opportunity to produce organisms tolerant to biotic or abiotic stress, in addition to exhibiting new shapes, colors and fragrances; this would be important in horticulture, where the proper selection of parents provides those advantages to the descendant hybrids. This review addresses the characteristics of each type of reproduction systems in orchids, as well as their advantages and disadvantages. At the same time, the study of the induction effect of metaxenia and xenia in this family is proposed. Finally, the production of orchid hybrids is contextualized and the opportunities of this approach in the near future.

Flower visits and pollinator pollen load networks reveal the effects of pollinator sharing on heterospecific pollen transfer in a subalpine plant community.

The mutualistic network of plant-pollinator also involves interspecific pollination caused by pollinator sharing. Plant-pollinator networks are commonly based on flower visit observations, which may not adequately represent the actual pollen transfer between co-flowering plant species. Here, we compared the network structure of plant-pollinator interactions based on flower visits (FV) and pollen loads (PL) on the bodies of pollinators and tested how the degree of pollinator sharing in the two networks affected heterospecific pollen transfer (HPT) between plant species in a subalpine meadow. The FV and PL networks were largely overlapped. PL network included more links than FV network. The positions of plant and pollinator species in the FV and PL networks were positively correlated, indicating that both networks could detect major plant-pollinator interactions. The degree of pollinator sharing, based on either the FV or the PL network, positively influenced the amount of heterospecific pollen transferred between plant species pairs. However, the degree of pollinator sharing had a low overall explanatory power for HPT, and the explanatory powers of the FV and PL networks were similar. Overall, our study highlights the importance of FV and PL for understanding the drivers and outcomes of plant-pollinator interactions, as well as their relevance to HPT.

FLORAL PHENOLOGY AND MORPHOMETRIC ANALYSIS OF THREE COMMERCIALLY GROWN COFFEA SPECIES

Coffea arabica, C. canephora, and C. liberica’s coexistence in one location or proximity areas could promote interspecific pollination. However, information related to floral phenology and flower structure of these three coffee species is insufficient. This research aimed to identify the divergence of flowering phenology and flower morphometrics among C. arabica, C. canephora, and C. liberica. Flowering phenology observations continued daily from July to October 2020, extending to two locations of parapatric populations of three coffee species. Flower morphometric measurements ensued at the peak period of flowering. The results showed some co-anthesis periods among the three coffee species, with C. liberica starting to flower early. Moreover, C. liberica var liberica had the broadest flower diameter and the highest petal number, whereas the tetraploid C. arabica had the lowest. Consequences of flowering phenology and flower structure on a pre-zygotic barrier of spontaneous, as well as, controlled interspecific hybridization among the three coffee species were the discussion focus.

Incomplete partitioning of pollinators by Linum suffruticosum and its coflowering congeners.

Linum suffruticosum shows variations in pollinator fit, pollen pickup, and local pollinators that predict pollen deposition rates. The species often coflowers with other Linum species using the same pollinators. We investigated whether L. suffruticosum trait variation could be explained by local patterns of pollinator sharing and associated evolution to reduce interspecific pollen transfer. Pollinator observations were made in different localities (single species, coflowering with other congeners). Floral traits were measured to detect differences across populations and from coflowering species. Reproductive costs were quantified using interspecific hand pollinations and measures of pollen-tube formation, combined with observations of pollen arrival on stigmas and pollen-tube formation after natural pollination in allopatric and sympatric localities. The size and identity of the most important pollinator of L. suffruticosum and whether there was pollinator sharing with coflowering species appeared to explain floral trait variation related to pollinator fit. The morphological overlap of the flowers of L. suffruticosum with those of coflowering species varied, depending on coflowering species identity. A post-pollination incompatibility system maintains reproductive isolation, but conspecific pollen-tube formation was lower after heterospecific pollination. Under natural pollination at sites of coflowering with congeners, conspecific pollen-tube formation was lower than at single-species localities. Trait variation in L. suffruticosum appears to respond to the most important local pollinator. Locally, incomplete pollinator partitioning might cause interspecific pollination, imposing reproductive costs. These reproductive costs may generate selection on floral traits for reduced morphological overlap with coflowering congeners, leading to the evolution of pollination ecotypes.

A sexual highly diploidized autotetraploid plant induced from the diploid cytotype of Paspalum chaseanum Parodi

AbstractPaspalum is one of the most common native grass genera of the New World grasslands, and it includes many wild natural forage species and rare domesticated ones. Polyploidy is widespread among the genus. Many species are multiploid, which basically involve two components: a sexual self‐incompatible diploid cytotype and a conspecific apomictic tetraploid, pseudogamous, and self‐compatible cytotype, though other apomictic polyploid levels may occur in the same species. Tetraploid individuals, reproducing exclusively sexually, have never been identified in a natural apomictic tetraploid Paspalum species. Doubling the chromosome number of a 2x cytotype is used to obtain a sexual autotetraploid to carry out sexual × apomictic crosses in breeding programs for plant improvement. After numerous attempts we generated only one autotetraploid plant of P. chaseanum (I4C) which reproduced sexually and was highly self‐incompatible. An unexpected preferential chromosome pairing resulted in the cytological diploidization of I4C. Despite each chromosome having three copies in I4C, meiotic chromosome configurations showed just 9.9% chromosomes forming multivalents (trivalents and quadrivalents), 82.1% bivalents, plus 7.9% univalents. The possible causes of this preferential pairing were analyzed. Although highly self‐incompatible, it was fertile in crosses following intra‐ and interspecific pollinations, but with variable seed set, from over 45% when crossed with an apomictic natural tetraploid strain of P. chaseanum, to 0.9% with a strain of P. wrightii. Thus, a novel autotetraploid P. chaseanum was synthesized with very interesting characteristics: sexual reproduction, high self‐incompatibility, variable cross‐compatibility, and high cytological diploidization. All valuable attributes for breeding programs in a forage grass.

Evaluation of pollen tube growth ability in Petunia species having different style lengths.

Pollen tube growth is essential for the fertilization process in angiosperms. When pollen grains arrive on the stigma, they germinate, and the pollen tubes elongate through the styles of the pistils to deliver sperm cells into the ovules to produce the seeds. The relationship between the growth rate and style length remains unclear. In previous studies, we developed a liquid pollen germination medium for observing pollen tube growth. In this study, using this medium, we examined the pollen tube growth ability in Petunia axillaris subsp. axillaris, P. axillaris subsp. parodii, P. integrifolia, and P. occidentalis, which have different style lengths. Petunia occidentalis had the longest pollen tubes after 6 h of culture but had a relatively shorter style. Conversely, the pollination experiments revealed that P. axillaris subsp. parodii, which had the longest style, produced the longest pollen tubes in vivo. The results revealed no clear relationship between the style lengths and the growth rate of pollen tubes in vitro. Interspecific pollinations indicated that the styles affected pollen tube growth. We concluded that, in vitro, the pollen tubes grow without being affected by the styles, whereas, in vivo, the styles significantly affected pollen tube growth. Furthermore, interspecific pollination experiments implied that the pollen tube growth tended to be suppressed in the styles of self-incompatibility species. Finally, we discussed the pollen tube growth ability in relation to style lengths.

Experimental sympatry suggests geographic isolation as an essential reproductive barrier between two sister species ofPedicularis

AbstractAlthough mechanical isolation mediated by shared pollinators has been considered as a classic model of pollinator‐mediated floral isolation inPedicularis, a superdiverse genus in Hengduan Mountains, southwest China, there has been no empirical study of interspecific pollen flow between closely related species. We examined reproductive barriers at six different stages betweenPedicularis cranolophaandPedicularis tricolor, two sister species. The two sister species were geographically isolated from each other based on our field survey and the records of herbarium specimens. Translocation experiments showed that flowering phenology partly overlapped and bumblebee pollinators did not discriminate between flowers of the two species. Bumblebee interspecific moves could mediate interspecific pollination as traced using fluorescent powder, in which pollen analogs placed on one species were transferred to the stigmas of the other species in experimental plots containing both species. Heterospecific pollen tubes grew in the style as well as conspecific pollen in hand‐pollination experiments. Reciprocal hybridization between the two species could produce (partially) viable seeds, suggesting weak post‐pollination barriers. Our results showed that geographic isolation was an important barrier of two species, and the total reproductive isolation between two species was incomplete when without geographical isolation. The formation of Big Snow Mountains could introduce an important pre‐zygotic reproductive barrier between the two sister species ofPedicularis; such geographical isolation could be responsible for allopatric speciation, giving a clue to understanding the rapid radiation on mountain areas.

Co-occurrence pattern of congeneric tree species provides conflicting evidence for competition relatedness hypothesis.

In plants, negative reproductive interaction among closely related species (i.e., reproductive interference) is known to hamper the coexistence of congeneric species while facilitation can increase species persistence. Since reproductive interference in plants may occur through interspecific pollination, the effective range of reproductive interference may reflects the spatial range of interspecific pollination. Therefore, we hypothesized that the coexistence of congeners on a small spatial scale would be less likely to occur by chance but that such coexistence would be likely to occur on a scale larger than interspecific pollination frequently occur. In the present study, we tested this hypothesis using spatially explicit woody plant survey data. Contrary to our prediction, congeneric tree species often coexisted at the finest spatial scale and significant exclusive distribution was not detected. Our results suggest that cooccurrence of congeneric tree species is not structured by reproductive interference, and they indicate the need for further research to explore the factors that mitigate the effects of reproductive interference.

Growing Environment and Heat Treatment Effects on Intra- and Interspecific Pollination in Chile Pepper (Capsicum spp.)

Heat tolerance is important for the sustainable production of many crops, including chile pepper. Tolerance to high temperature is complex and involves various component traits, with pollen viability being among the most important. in vitro pollen assays for heat tolerance have been widely used in chile pepper; however, associations between the pollen treatment and pollination have not been widely explored. The objectives of this study were to validate the utility of in vitro heat stress pollen characterization through in vivo pollination during summer and winter seasons and to evaluate the cross-compatibility among wild and domesticated species to initiate introgression population development. Seven entries of wild and domestic Capsicum species grown during the summer and winter seasons were used to evaluate pollination success rate. Pollen was either used directly or treated at 38 °C for four hours before making reciprocal self- and cross-pollination among all the entries. Significant associations between in vitro pollen treatment and pollination success rate during summer and winter seasons were identified. Heat treatment was a greater contributor to variability than the growing environment, which validates previous reports on the usefulness of studying pollen in vitro in selection for heat tolerance. Accessions of the wild progenitor C. annuum var glabriusculum, PBC 1969 and PBC 1970, were identified as a potential heat-tolerant source for use in breeding and future research. This work provides a basis for future research in exploring additional heat tolerance components as well as for the development of phenotyping assays for pollen or other floral traits.

Pollinator sharing among co-flowering plants mediates patterns of pollen transfer

Co-flowering plant species often share pollinators, which could result in interspecific pollination. Despite some evidence suggesting plant species with overlapping pollinators influence each other’s pollination, the relationship between pollinator sharing and heterospecific pollen transfer (HPT) is not well explained at the community level. Here, we sampled a plant-pollinator visitation network to calculate the degree of pollinator sharing, and we identified pollen deposition on stigmas of co-flowering species to construct a HPT network in a sub-alpine meadow in southwest China. We also analyzed floral colour using reflectance spectra to measure the flower reflectance dissimilarity between each pair species. We found that there was no pollen transfer between most species pairs with shared pollinators. A higher proportion of HPT links between plant species pairs with shared pollinators was observed than between pairs with distinct pollinators. More pollen grains were transferred from donor to recipient species with greater pollinator sharing. Flowers with more dissimilar flower colours exchanged less heterospecific pollen. Our study evaluated the effect of pollinator sharing in shaping pollen transfer patterns among co-flowering species, which may shed light on the ecological implications of plant–pollinator interactions, mediated by pollinator sharing.

Phenological shift along an elevational gradient and dispersal of pollen and seeds maintain a hybrid zone between two cherry tree species

AbstractHybrid zones between species with staggered reproductive phenology sometimes occur along elevational gradients. The maintenance of such hybrid zones may depend on elevational shift in phenology and vertical dispersal of pollen and seeds. In a hybrid zone of Cerasus leveilleana (Cl) and C. sargentii (Cs), 111 adults, 372 embryos of 65 mothers, and 133 juveniles were sampled across elevations of 900–1,400 m in central Japan. Using the hybrid index estimated from nuclear microsatellites, the samples were assigned to Cl, Cs or their hybrid taxon. Cs was in higher elevations than Cl with an overlapping range, and their hybrids were in intermediary elevations. Flowering periods were earlier in Cs than in Cl with little overlap at the same elevation and were intermediate in their hybrids. Flowering periods were delayed in higher elevations and overlapped between Cl and Cs in different elevations. Fruiting periods were slightly earlier in Cs than in Cl but were largely overlapped. Effective pollinators and seed dispersers were common among the taxa. Changes in the hybrid index from mothers to their embryos were larger in hybrid mothers than in Cl or Cs mothers, indicating mating of hybrids with diverse taxa. In Cs, juveniles were more abundant in lower elevations than embryos, indicating downhill seed dispersal. Proportions of hybrids were 19% in embryos, 17% in juveniles and 12% in adults. The findings suggest that the hybrid zone is maintained by interspecific pollination between different elevations, backcrossing with hybrids at similar elevations, vertical seed dispersal and mild selection against hybrids.

Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum.

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.

A hybridization technique for orphan legumes: development of an artificial interspecific pollination protocol for Crotalaria spp.

ABSTRACT Crotalaria brevidens and C. ochroleuca commonly grown as vegetables have been extensively studied. However, information on breeding techniques is scanty. This study aimed to develop an artificial interspecific pollination protocol for two genotypes of Crotalaria with contrasting leaf and pod morphological features. The two are landraces that were previously collected from two counties in Kenya. The experimental design was a completely randomized design (CRD) in a greenhouse with C. ochroleuca as the female parent and C. brevidens as the male parent. Six-day-old flower buds of the female parent were emasculated. Pollen from a freshly opened flower was rubbed over the stigma of the emasculated flower. The pollinated stigma was inserted back into the keel petal and covered by the wing and standard petal. Data on success rate, pod, and seed production were subjected to analysis of variance. The developed artificial pollination method showed 75% success rate in the interspecies cross of C. ochroleuca and C. brevidens. Time of crossing did not significantly influence the success rate, pod, and seed production. The study demonstrated artificial interspecific pollination of Crotalaria by keel petal incision rubbing method. This protocol lays the foundation for genetic studies and improvement of Crotalaria spp.

Floral biology and potential hybridization of three sympatric epiphytic bromeliads in Veracruz, Mexico

AbstractIn the Bromeliaceae, hybrids have been engineered for horticultural purposes; in addition, hybrids have been observed in natural habitats. We investigated the hybridization potential of three sympatric species of Tillandsia inhabiting secondary forests and shaded coffee plantations in the central mountains of Veracruz, Mexico. The studied species (T. juncea, T. polystachia and T. variabilis) show overlap in flowering phenology and are believed to share pollinators. We compared the floral morphology of the species, performed controlled intra‐ and interspecific pollinations, and recorded pollen tube growth, fruit and seed set, as well as seed germination. Flower size differed among the species, but stamen and stigma heights were similar between pairs of species, which would facilitate interspecific pollen exchange. Pollen tubes reached the ovary in interspecific crosses. The three species showed interspecific cross‐compatibility, as all crosses resulted in development of mature fruit and viable seeds. Tillandsia juncea achieved the highest fruit set, whereas T. variabilis showed the highest seed set. Seed set as a result of agamospermy, autogamy, geitonogamy and interspecific cross‐pollination did not differ among the species. The findings suggest that pre‐ and post‐pollination barriers are weak among the three species of Tillandsia, which thus could potentially hybridize in natural sympatric populations.

Photosynthetic Mechanisms of Metaxenia Responsible for Enlargement of Carya cathayensis Fruits at Late Growth Stages.

Fruits of hickory (Carya cathayensis) are larger and their peel is greener after interspecific pollination by pecan (Carya illinoinensis; later pp fruits) than after intraspecific pollination by hickory (later ph fruits). Previous studies have found little genetic differences between offspring and their maternal parent, indicating that the observed trait differences between pp and ph fruits are due to metaxenia. Fruit development depends on the amount of photosynthetic assimilate available. Since there is no difference in photosynthesis of the associated leaves between pp and ph fruits, the larger size of the pp fruits might be attributed to changes in fruit photosynthesis caused by the different pollen sources. To elucidate to the photosynthetic mechanisms behind the metaxenia effect on fruit development in hickory, the effects of intraspecific and interspecific pollination regimes were examined in the present study. We observed the photosynthetic capacity in the peel of fruits and the related ecophysiological and morphological traits of both ph and pp fruits over a period of 120 days after pollination. Significant differences in the appearance and dry weight between ph and pp fruits were observed at 50 days after pollination (DAP). More than 70% of dry matter accumulation of the fruits was completed during 60–120 DAP, while the true photosynthetic rate of the associated leaves significantly decreased by about 50% during the same period. In several cell layers of the peel, the number of chloroplasts per cell was significantly higher in pp than in ph fruits. Similarly, the ribulose 1, 5-bisphosphate carboxylase (Rubisco) activity, the total chlorophyll content, and the nitrogen content were all significantly higher in pp than in ph fruits during all growth stages; and all of these physiological quantities were positively correlated with the gross photosynthetic rate of the fruits. We conclude that the enhanced photosynthetic capacity of pp fruits contributes to their fast dry matter accumulation and oil formation. This result will provide a theoretical basis for improving hickory fruit yields in practical cultivation.

Multi‐year stigmatic pollen‐load sampling reveals temporal stability in interspecific pollination of flowers in a subalpine meadow

Co‐flowering plants may commonly experience interspecific pollination. It remains unknown, however, whether interspecific pollination is a largely stochastic process or consistent enough over years to exert selection for traits that can reduce interspecific pollination or ameliorate its deleterious effects on reproduction. To assess the likelihood of this precondition being met, stigmatic pollen loads on 17–34 insect‐pollinated plant species over three consecutive years were scored in a subalpine meadow in southwestern China. Plant species varied significantly in the amount and proportion of heterospecific pollen (HP) on stigmas. Both the number of HP species and the proportion of the pollen load that was HP for each recipient species correlated positively between years (reflected in pairwise correlations for all year‐by‐year combinations). Although inter‐annual variation was smaller for conspecific pollen (CP) than for HP loads, species tended to experience either consistently high or consistently low HP proportions across years. We found that species with higher stigmatic HP proportions generally experienced lower proportional variation in stigmatic HP, an unexpected result if high HP loads are the result of rare stochastic events. The novel finding of between‐year consistency in stigmatic loads of heterospecific pollen suggests that adaption to stigmatic loads of HP is possible, and two divergent strategies may have evolved: HP avoidance and HP tolerance. The observation of temporally consistent differences among species in levels of HP supports the idea that natural selection may be operating either to increase tolerance or to minimize arrival of heterospecific pollen on stigmas in co‐flowering plants. Such adaptations may be important for the maintenance of high levels of local plant diversity in biodiversity hotspots such as our study area.

Taxonomic and functional diversity of the co‐flowering community differentially affect Cakile edentula pollination at different spatial scales

Abstract The number of co‐flowering species, floral density and floral trait diversity can be major determinants of pollinator‐mediated plant–plant interactions in a community. However, evaluation of how each one of these co‐flowering components affects the pollination success of a single focal plant species, and how these effects vary at different spatial scales, is lacking. Here, we evaluated the effects of functional diversity (flower morphology and colour), taxonomic diversity (reflecting potential sampling effects) and flower density (conspecific and heterospecific), on the pollinator environment (i.e. visitation rate and pollinator diversity) and pollination success (i.e. pollen load size and number of pollen tubes per style) of Cakile edentula (Brassicaceae). We applied structural equation models (SEMs) at the floral‐neighbourhood (plot level) and community‐wide scales to uncover the factors that mediate co‐flowering community effects on C. edentula pollination success. We found that co‐flowering community effects at the community level are more important than fine‐scale floral‐neighbourhood differences in mediating plant pollination success in our study species. Increasing plant functional diversity decreased pollinator visitation rate but increased the diversity of pollinator functional groups visiting C. edentula flowers. Taxonomic diversity negatively affected pollinator diversity suggesting that other unmeasured floral traits may be relevant or that single‐species effects (sampling effects) may be important. Overall, our results suggest that functional floral trait diversity in a community may be the most important factor influencing pollination success of species in a community. We also found evidence for intra‐ and interspecific pollinator competition mediated by flower density, but none of these effects seemed to have a significant impact on pollination success. This study is an important step towards understanding the complexity of co‐flowering community effects on the pollination success of individual plant species at multiple spatial scales. This study further reveals the potential importance of plant functional diversity in a community in helping predict competitive and facilitative interactions in co‐flowering communities. Synthesis. Floral density and taxonomic and functional co‐flowering diversity are important drivers of pollination success in flowering plants. The effects of the co‐flowering diversity on the pollination success of plant species can largely depend on the spatial scale being studied. Only evaluating the outcomes of pollinator‐mediated plant–plant interactions at multiple stages of the pollination process can lead to a complete understanding of their ecological consequences in nature.

Fruit set and plant regeneration in cassava following interspecific pollination with castor bean

The increasing demand for cassava (Manihot esculenta Crantz) for food and non-food uses in the tropics necessitates that its breeding for increased root productivity be made faster. The characteristic long breeding cycle and heterozygous nature of this crop, pose a major obstacle to its rapid genetic improvement. This study aimed at inter-pollinating cassava with castor bean (Ricinus communis), with a purpose of inducing and regenerating cassava doubled haploids (DHs). A total of 3,349 flowers from twelve elite cassava varieties were inter-pollinated with caster bean. A total of 803 fruits were harvested for early embryo rescue and/or ovule culture. Of these, three were dissected to obtain seven unique embryos, while 800 were dissected to obtain 1312 young ovules, all of which were cultured in vitro. Overall, 82 (6.25%) of the cultured ovules formed callus that originated from the embryosac region, which is haploid. Four out of seven rescued embryos (57.1%) regenerated into plantlets. Ploidy analyses of 24 samples using flow cytometry revealed that 23 of the analysed samples were diploid. However, one callus sample was anueploid. Only one sample had an exceptionally high level of homozygosity ( 84.2%). These findings lay a foundation for future research aimed at induction of haploids in cassava.Key words: Doubled haploid, embryo rescue, ovule culture, ploidy

Reproductive biology of two synchronopatric neotropical species of Epidendrum (Orchidaceae)

The reproductive system of Orchidaceae is predominantly xenogamous and highly dependent on animal pollen vectors. Nectar is the main floral resource, offered to pollinators in perigonal nectaries, cuniculi or spurs; these structures are often difficult to locate and to evaluate their functionality. The Neotropical Epidendrum denticulatum Barb. Rodr. and Epidendrum orchidiflorum Salzm. ex. Lindl. bloom synchronously throughout the year in the municipality of Maricá (Rio de Janeiro State, SE Brazil). The flowers of E. denticulatum open in the morning, have a conspicuous pink colour, a nectar guide, and nuptial and extranuptial nectaries. During the day, they are visited by species of Hesperiidae (Lepidoptera), which remove the pollinia. The flowers of E. orchidiflorum open at night, are green with nuptial and extranuptial nectaries, and have a strong odour at night, suggesting nocturnal pollination agents, possibly moths. To date, no information is available on the reproductive biology and pollination mechanisms for E. orchidiflorum, nor is micromorphological, anatomical or histochemical data related to the presence and functionality of secretory structures associated with pollination in either species. Also, no studies have examined the occurrence of mechanisms that hinder or prevent interspecific pollination. Here, through reproductive, morphological, structural and histochemical analyses, we evaluated the floral biology and reproductive systems of these synchronopatric Epidendrum species. Both species have extranuptial nectaries, a functional cuniculus, and osmophores. Hand-pollination experiments revealed that the species are self- and intercompatible, although individuals with intermediate morphology were not identified. Pre-pollination barriers related to the floral biology of the species seem to be sufficient to prevent hybridization, but other biological aspects certainly contribute to the genetic integrity of the populations, such as the low rates of visits to flowers, low percentage of fruit and seed set, low seedling recruitment, and high investment in vegetative propagation.

In vivo interspecific pollination success between Pinus radiata, P. maximinoi, P. oocarpa and P. tecunumanii

The objective of the study was to investigate in vivo interspecific pollination success between Pinus radiata, P. maximinoi, P. oocarpa and P. tecunumanii. Pinus radiata was control pollinated with pollen lots of P. maximinoi, P. oocarpa and P. tecunumanii in a P. radiata seed orchard at Karatara (Southern Cape, South Africa). Pollination success was determined by counting the number of visible ovules, pollen grains inside and outside P. radiata ovules, as well as pollen tubes visible inside P. radiata ovules. Conelets were harvested and studied at eight time intervals, including 24 h after pollination, and weekly for 7 weeks after pollination. Histology studies with a standard fixation-dehydration-embedding sequence and paraffin wax method were used to determine the number of visible pollen grains inside versus outside the ovules and number of pollen tubes. Results indicated that pollen grains did sift through the cone scales within 24 h after pollination. However, P. radiata differed significantly (time by type of cross interaction) from the other three hybrid combinations in terms of number of visible ovules, visible pollen grains inside and outside of the ovules as well as pollen tubes, confirming limited interspecific hybridisation success. Future studies need to determine the percentage of fertile ovules in cross combination as a tool in predicting pollination success.