Sílvia Castro

Effect of pollination on floral longevity and costs of delaying fertilization in the out-crossing Polygala vayredae Costa (Polygalaceae).

BACKGROUND AND AIMS The effect of pollination on flower life span has been widely studied, but so far little attention has been paid to the reproductive consequences of delayed pollination in plants with long floral life spans. In the present study, Polygala vayredae was used to answer the following questions. (1) How does male and female success affect the floral longevity of individual flowers? (2) How does delaying fertilization affect the female fitness of this species? METHODS Floral longevity was studied after experimental pollinations involving male and/or female accomplishment, bagging and open pollination. The reproductive costs of a delay in the moment of fertilization were evaluated through fruit set, seed-ovule ratio and seed weight, after pollination of flowers that had been bagged for 2-18 d. KEY RESULTS Senescence of the flowers of P. vayredae was activated by pollen reception on the stigmatic papillae, while pollen removal had no effect on floral longevity. Nonetheless, a minimum longevity of 8 d was detected, even after successful pollination and pollen dissemination. This period may be involved with the enhancement of male accrual rates, as the female accomplishment is generally achieved after the first visit. Floral life span of open-pollinated flowers was variable and negatively correlated with pollinator visitation rates. Delayed pollination had a major impact on the reproductive success of the plant, with fruit set, seed-ovule ratio and seed weight being significantly diminished with the increase of flower age at the moment of fertilization. CONCLUSIONS A strong relationship between pollination and floral longevity was observed. Flowers revealed the ability to extend or reduce their longevity, within some limits, in response to the abundance of efficient pollinators (i.e. reproductive fulfilment rates). Furthermore, with scarce or unpredictable pollinators, a long floral life span could maintain the opportunity for fertilization but would also have reproductive costs on production of offspring. Reduced female fitness late in the flowers life could shift the cost-benefit balance towards a shorter life span, partially counteracting the selection for longer floral life span potentially mediated by scarce pollination services.

Flow cytometric and cytogenetic analyses of Iberian Peninsula Festuca spp.

Festuca L. has an important diversification centre in the Iberian Peninsula. We used chromosome counting, fluorescence (FISH) and genomic in situ hybridization (GISH), and DNA flow cytometry (FCM) to clarify the taxonomic position of several taxa, to search for phylogenetic relationships and to assess the extent and pattern of genome variation in fescues. The chromosome number of Festuca duriotagana var. barbata is determined for the first time and new ploidy level estimations are given for F. rothmaleri and F. summilusitana. In the latter species, besides the reported decaploid level, dodecaploidy was found in some populations, which points to the existence of an unrecognized taxon. Moreover, these differences were confirmed by FCM and a high positive correlation was found with the type of substrate where F. summilusitana was growing. For each section, a decrease of genome size with increase of polyploidy was observed. In general, in situ hybridization techniques failed to reveal phylogenetic relationships among the selected species. In FISH, a variation in the number of rDNA sites was observed in some species. GISH results indicate that F. henriquesii is not a progenitor of the studied polyploid species.

Consequences of nectar robbing for the fitness of a threatened plant species

The effect of nectar robbing on plant fitness is poorly understood and restricted to a few plant species. Furthermore, the available studies generally evaluate the effects of nectar robbing on female fitness, disregarding the male component. Here we measured the effects of the nectar-robbing bumblebees on male (measured as pollen analogue flow distance) and female (measured as seed production) reproductive success in the insect-dependent Polygala vayredae, a narrow endemic species from the pre-Pyrenees (Spain). Intense nectar robbing by bumblebees significantly reduced the nectar available to legitimate pollinators in the studied population, and this reduction affected both male and female fitness. Significant differences were observed in fluorescent dye dispersion between robbed and non-robbed flowers within the population. Fluorescent dyes from non-robbed flowers were dispersed to larger distances and over a larger number of flowers when compared with robbed ones. Moreover, significant differences were observed in both fruit set and seed ovule ratios between the two groups, with non-robbed flowers presenting higher reproductive outcomes. However, no effect on seed weight was detected among treatments. The data obtained suggest that in this species, nectar robbing has important indirect and negative effects on plant fecundity, through both male and female functions, due to a modification in the foraging behaviour of legitimate visitors.

Somatic embryogenesis in leaf callus from a mature Quercus suber L. Tree

SummarySomatic embryos were obtained from a 60-yr-old Quercus suber L. tree. Leaf explants were cultivated on Murashige and Skoog medium with 30 gl−1 sucrose, 3 gl−1 gelrite, pH adjusted to 5.8, and different growth regulator combinations. Callus induction took place at 24±1°C in the dark during the first 3 wk. After 3 mo, calluses that showed embryogenic structures were transferred to the same medium without growth regulators. Somatic embryogenesis was only observed in calluses induced on E3 medium (supplemented with 4.5 μM 2,4-dichlorophenoxyacetic acid and 9.0 μM zeatin). On average, 7.5% of the initial explants formed embryogenic calluses in this medium. Somatic embryo proliferation was high due to secondary embryogenesis. On average, 10% of the somatic embryos germinated and 40% of these germinated embryos converted into plants. Plants were elongated on the same medium without growth regulators and acclimated to greenhouse conditions.

Stigma–anther reciprocity, pollinators, and pollen transfer efficiency in populations of heterostylous species of Lithodora and Glandora (Boraginaceae)

According to Darwin, the reciprocal position of sexual whorls in heterostylous plants enhances disassortative pollen transfer between different floral morphs. It is believed that greater reciprocity between morphs will promote more efficient transfer of pollen. Additionally, efficient pollination will act as a selective force in achieving greater reciprocity between floral morphs. In this study we test whether variation in reciprocity of sexual organs between morphs is related to the efficiency of pollinators in transferring pollen between them. To do this, we first describe the pollinator’s array in several populations of species of the genus formerly known as Lithodora, which have different types of stylar polymorphism and degrees of reciprocity, and determine their abundance, plant visitation rate, number of flowers visited per plant and handling time in the population. We estimate the efficiency of the pollinator arrays by use of an approximation based on qualitative (location of pollen loads on different areas of insect bodies) and quantitative (plant visitation rate) measurements. Our results show a correlation between the degree of reciprocity and the efficiency of pollinators associated with the populations. These observations suggest that pollinators are a possible selective force driving the evolution of heterostyly.

How does secondary pollen presentation affect the fitness of Polygala vayredae (Polygalaceae)

Secondary pollen presentation is the relocation and presentation of pollen in floral structures (termed pollen presenters) other than the anthers. These pollen presenters are often found close to the stigma and have been hypothesized to increase the accuracy of pollen transfer, although no experimental studies have been done. We examined the function of the pollen presenter and its efficiency in pollen dispersal, female fitness, and the degree of interference created by self-pollen in the shrublet Polygala vayredae, an insect-pollinated species with secondary pollen presentation. Herkogamy, a mechanism generally involved in the reduction of self-interference, was also evaluated. Significant pollen was lost (49% of total pollen) during the secondary relocation in the pollen presenter. However, pollen was exported from the pollen presenter, and subsequent pollen losses were similar to those in species with primary pollen presentation. Despite the presence of a self-incompatibility system, the numbers of developed pollen tubes as well as fruit and seed production were significantly reduced by the self-pollen interference created at the stigmatic papillae level. The extent of herkogamy correlated positively with female fitness. The secondary pollen presentation mechanism may in fact be an accurate system for pollen transport, but it may also have its costs. Further comparative studies involving species with primary and secondary pollen presentation are needed to fully understand the advantages and disadvantages of secondary pollen presentation.

Effect of invader removal: pollinators stay but some native plants miss their new friend.

Removal of invasive species often benefits biological diversity allowing ecosystems’ recovery. However, it is important to assess the functional roles that invaders may have established in their new areas to avoid unexpected results from species elimination. Invasive animal-pollinated plants may affect the plant–pollination interactions by changing pollinator availability and/or behaviour in the community. Thus, removal of an invasive plant may have important effects on pollinator community that may then be reflected positive or negatively on the reproductive success of native plants. The objective of this study was to assess the effect of removing Oxalis pes-caprae, an invasive weed widely spread in the Mediterranean basin, on plant–pollinator interactions and on the reproductive success of co-flowering native plants. For this, a disturbed area in central Portugal, where this species is highly abundant, was selected. Visitation rates, natural pollen loads, pollen tube growth and natural fruit set of native plants were compared in the presence of O. pes-caprae and after manual removal of their flowers. Our results showed a highly resilient pollination network but also revealed some facilitative effects of O. pes-caprae on the reproductive success of co-flowering native plants. Reproductive success of the native plants seems to depend not only on the number and diversity of floral visitors, but also on their efficiency as pollinators. The information provided on the effects of invasive species on the sexual reproductive success of natives is essential for adequate management of invaded areas.

Physical distribution of homoeologous recombination in individual chromosomes of Festuca pratensis in Lolium multiflorum.

Crossing over-based recombination is a powerful tool for generating new allelic combinations during sexual reproduction. It usually occurs between homologous chromosomes. However, under some conditions, homoeologues may also be capable of crossing over. Whether homologous and homoeologous crossovers are equivalent and governed by the same rules has never been established. Here we report on chromosome distribution of homoeologous crossovers in a unique system of Festuca × Lolium hybrids. Unlike in most other hybrids, in these intergeneric hybrids, homoeologous chromosomes are capable of pairing and crossing over with frequencies approaching that of homologues. At the same time, genome divergence makes cytological detection of chromosome recombination feasible. We analyzed the distribution of homoeologous recombination along individual chromosomes in a complete set of intergeneric single chromosome substitutions fromF. pratensisinto tetraploid L. multiflorum. Homoeologous recombination sites were not evenly distributed along the chromosomes, being concentrated in intercalary regions of the arms and reduced in proximal and distal regions. Several recombination hotspots and cold spots were found along individual chromosomes and the recombination was not affected by the presence of a secondary constriction. Our results indicate that despite the uneven distribution of homoeologous recombination, introgression of any part of the F. pratensis genome into L. multiflorum is feasible.

Absence of gene flow between diploids and hexaploids of Aster amellus at multiple spatial scales

The potential for gene exchange across ploidy levels has long been recognized, but only a few studies have explored the rate of gene flow among different cytotypes. In addition, most of the existing knowledge comes from contact zones between diploids and tetraploids. The purpose of this paper was to investigate relationships between diploid and hexaploid individuals within the Aster amellus aggregate. A. amellus is known to occur in diploid and hexaploid cytotypes in Europe, with a complex contact zone in central Europe. Patterns of genetic diversity were investigated using seven microsatellite loci at three different spatial scales: (1) in the single known mixed-ploidy population; (2) in populations at the contact zone and (3) in a wider range of populations across Europe. The results show clear separation of the cytotypes at all three spatial scales. In addition, analysis of molecular variance strongly supported a model predicting a single origin of the hexaploids, with no or very limited gene flow between the cytotypes. Some hexaploid individuals found in the mixed-ploidy population, however, fell into the diploid cluster. This could suggest recurrent polyploid formation or occasional cross-pollination between cytotypes; however, there are strong post-zygotic breeding barriers between the two cytotypes, making the latter less plausible. Overall, the results suggest that the cytotypes could represent two cryptic species. Nevertheless, their formal separation is difficult as they cannot be distinguished morphologically, occupy very similar habitat conditions and have largely overlapping distribution ranges. These results show that polyploid complexes must be treated with caution as they can hide biological diversity and can have different adaptation potentials, evolving independently.

Unusual heterostyly: style dimorphism and self-incompatibility are not tightly associated in Lithodora and Glandora (Boraginaceae)

BACKGROUND AND AIMS Heterostyly is a floral polymorphism characterized by the reciprocal position of stamens and stigmas in different flower morphs in a population. This reciprocal herkogamy is usually associated with an incompatibility system that prevents selfing and intra-morph fertilization, termed a heteromorphic incompatibility system. In different evolutionary models explaining heterostyly, it has been alternately argued that heteromorphic incompatibility either preceded or followed the evolution of reciprocal herkogamy. In some models, reciprocal herkogamy and incompatibility have been hypothesized to be linked together during the evolution of the heterostylous system. METHODS We examine the incompatibility systems in species with different stylar polymorphisms from the genera Lithodora and Glandora (Boraginaceae). We then test whether evolution towards reciprocal herkogamy is associated with the acquisition of incompatibility. To this end, a phylogeny of these genera and related species is reconstructed and the morphological and reproductive changes that occurred during the course of evolution are assessed. KEY RESULTS Both self-compatibility and self-incompatibility are found within the studied genera, along with different degrees of intra-morph compatibility. We report for the first time extensive variability among members of the genus Glandora and related species in terms of the presence or absence of intraspecies polymorphism and heteromorphic incompatibility. Overall, our results do not support a tight link between floral polymorphism and incompatibility systems. CONCLUSIONS The independent evolution of stylar polymorphism and incompatibility appears to have occurred in this group of plants. This refutes the canonical view that there is strong linkage between these reproductive traits.