The potential for selection on pollen colour dimorphisms in Nigella degenii: morph-specific differences in pollinator visitation, fertilisation success and siring ability

Two subspecies of Nigella degenii (Ranunculaceae) possess a dimorphism in pollen colour and vary extensively in frequency of the two morphs in natural populations. Here we investigate the role of selection on pollen colour during the pollination phase in the two subspecies and its potential contribution to the maintenance of this colour variation. In a combination of common garden experiments and field observations, we obtained data on pollinator visitation rates and explored the effect of pollen colour on fertilisation success and siring ability under conditions of low vs. high pollen competition. In experimental gardens, naïve pollinators responded differently to plants with different pollen colour, but the favoured morph varied between dates and locations, and colour morphs were not visited in a frequency-dependent manner. Donor plants with dark pollen had a reproductive advantage (higher seed set) in single-donor pollinations, but the realised siring ability (measured by progeny morph ratio) was highly variable between different two-donor crosses with no general bias towards the light or dark morph. Therefore, although the dark pollen type appears to have a general selective advantage in terms of fertilisation success, our data are also consistent with a scenario involving the maintenance of both colour morphs, particularly under conditions of high pollen competition, a variable genetic background and/or spatial or temporal variation in the pollinator fauna.     

Evolution and maintenance of pollen-colour dimorphisms in Nigella degenii: habitat-correlated variation and morph-by-environment interactions

Dimorphism in pollen colour is rare among flowering plants, but occurs in two geographically and morphologically distinct subspecies of Nigella degenii (Ranunculaceae). We evaluated the role of genotype-by-environment interactions in the maintenance of two pollen morphs within each of these subspecies. Morph frequencies in a number of populations were related to current habitat conditions, and an extensive common-garden experiment involving both optimal and stressful conditions (drought and nutrient deficiency) was carried out. The putatively derived (dark) pollen morph of N. degenii ssp. barbro has a higher frequency on slopes facing north or east than on slopes facing south or west. Plants of the dark morph also have a higher mortality under drought stress or nutrient deficiency. Data available for N. degenii ssp. jenny provide little evidence for habitat-correlated variation in morph frequency or morph-specific differences in fitness under optimal and stressful growth conditions. Our results suggest that morph-by-environment interactions in mortality could contribute to the maintenance of pollen-colour dimorphisms in N. degenii ssp. barbro.     

Sex-allocation trade-offs in Nigella sativa (Ranunculaceae) examined with flower manipulation experiments

Sex-allocation trade-offs have long been invoked as a primary factor underlying the evolution of separate sexes and the reduction of pollen production accompanying the evolution of selfing. In the present study, I conducted stamen and style removal experiments to explore the existence of such trade-offs in Nigella sativa, a hermaphroditic plant species whose flower structure allows early manipulation of both male and female function. Plants on which all stamens were removed at the bud stage had a higher rate of flower initiation and produced significantly heavier seeds than did plants whose flowers remained intact, apparently by using resources that were released when the stamens were removed. However, there was no effect of stamen removal on the number of flowers that reached anthesis, the total biomass allocated to seed production, or the vigour of plants in the progeny generation. In contrast, prevention of fruit production (style removal) increased the amount of biomass invested in stamen by 57% relative to plants whose flowers were allowed to set fruit. These observations verify the existence of a sexual trade-off in N. sativa but also raise the possibility that stamen-suppressing mutations sometimes lack the pleiotropic consequences of increasing female function, at least in species with large, expensive fruits.     

Floral costs in Nigella sativa (Ranunculaceae): compensatory responses to perianth removal

Because internal resources are finite, it has been assumed that attractive, floral organs represent a significant drain on the energy and nutrient budget of a plant. Despite the broad significance of such trade-offs, in relatively few studies have investigators manipulated floral investments, then evaluated allocation to subsequently produced flowers, fruits, and seeds. In the present study of Nigella sativa, the cost of maturing and/or maintaining perianths was documented after all sepals and nectaries were removed at the bud stage and a significant increase in mean seed mass, the total amount of biomass allocated to seed production, and mean germination rate of the maternal seed crop were measured. The increased biomass, carbon, and nitrogen allocated to seeds were similar in magnitude to the reduction in biomass, carbon, and nitrogen invested in sepals and nectaries after perianth removal. Perianth removal did not significantly affect flower production, maternal fecundity, or progeny seed number. Taken together, these observations indicate the potential for selection-mediated through resource trade-offs with seed mass and time to germination-to cause, or at least facilitate, evolutionary reductions in flower size.     

The oldest evidence of Nigella damascena L. (Ranunculaceae) and its possible introduction to central Europe

After the beginning of metal processing at the transition from the Neolithic to the Bronze Age, further knowledge of ore mining and smelting had spread from the Near East to central Europe. In the copper ore deposits of Schwaz, in the central part of the Alps, the oldest traces of copper mining derive from the early to middle Bronze Ages. Investigation of a middle to late Bronze Age (1410–920 cal B.C.) slag-washing site in the area revealed a carbonised seed of Nigella damascena (Ranunculaceae) (love-in-a-mist) together with individual other food plants. The plant remains had become incorporated into the slag sediments by chance and had been preserved in an excellent state due to toxic copper salts contained in the soil. Nigella damascena, like N. sativa (black cumin), is traditionally used as a condiment and healing herb in southern Europe and the Near East, but has never grown in the wild in central Europe. Until now, there has been no evidence of prehistoric large-scale cultivation of N. damascena in central Europe. This leads to two possible conclusions: the find may either originate from an exchange of goods with the cultures in the Mediterranean during the Bronze Age, or indicate an introduction of the plant by an immigrant population from that area. Implicating the latter alternative together with the archaeological context of the ore processing site suggests that Nigella damascena had been introduced to the Alps by foreign miners in the course of ore exploitation during the middle to late Bronze Age.     

A naturally occurring chromosomal translocation in Myzus persicae (Sulzer)

Populations of M.persicae from glasshouses in Britain were found to have a structurally heterozygous chromosome complement. Biometric data from somatic metaphase chromosomes, and the pairing configurations of chromosomes at spermatocyte meiosis, indicate either a simple or a reciprocal translocation involving autosomes 1 and 3. All except one of the clones started from these populations showed high levels of esterase activity indicative of resistance to organophosphorus insecticides. The British translocated clones were all androcyclic in reproductive character, but of several distinct genotypes. A similar or identical translocation was found in seven out of nine clones started from M.persicae collected in the field in Japan. The translocation was present in Japanese M.persicae of all reproductive categories, being somehow maintained in the sexual breeding system of the aphid. In breeding experiments, translocation heterozygosity was inherited independently from colour and reproductive characters. The translocation was also found in a sample of M.persicae from southern California.     

Variation of pollen morphology, and its implications in the phylogeny of Clematis (Ranunculaceae)

Clematis s.l. (including Archiclematis and Naravelia) is a genus of approximately 300 species with cosmopolitan distribution. The diversity of its pollen was surveyed in 162 taxa belonging to all infrageneric groups of Clematis s.l. Pollen morphology was investigated by use of scanning electron microscopy to identify useful characters, test taxonomic and systematic hypotheses, and elucidate pollen character evolution on the basis of the molecular phylogeny. Clematis pollen is small to medium (14.8–32.1?μm?×?14.2–28.7?μm), oblate to prolate (P/E?=?0.9–1.4) in shape. The apertures may be tricolpate and pantoporate sometimes with 4-zonocolpate and pantocolpate pollen grains as transitional forms. The tricolpate pollen grains are predominant and occur in all the sections of the genus, whereas pantoporate pollen grains can be found in sect. Tubulosae, sect. Viorna, sect. Viticella, and Naravelia only. Phylogenetic mapping of aperture types reveals that the pantoporate pollen type may be the apomorphy in the genus and evolved several times. The surface ornamentation in all taxa studied is similar and characterized by microechinae evenly distributed on the microperforate tectum. The size and density of spinules on the tectum vary greatly but successive in the whole genus. According to the character syndromes of the ornamentation, separating sect. Brachiata from sect. Meclatis is supported. Though pollen morphology may contribute to investigation of problematic taxa, the taxonomic value of pollen morphology is limited at the species level.     

Functional characterization of naturally occurring genetic variations of the human guanine-rich RNA sequence binding factor 1 (GRSF1)

The guanine-rich RNA sequence binding factor 1 (GRSF1) constitutes an ubiquitously occurring RNA-binding protein (RBP), which belongs to the family of heterogeneous nuclear ribonucleoprotein F/H (hnRNP F/H). It has been implicated in nuclear, cytosolic and mitochondrial RNA metabolism. Although the crystal structures of GRSF1 orthologs have not been solved, amino acid alignments with similar RNA-binding proteins suggested the existence of three RNA-binding domains designated quasi-RNA recognition motifs (qRRMs). Here we established 3D–models for the three qRRMs of human GRSF1 on the basis of the NMR structure of hnRNP F and identified the putative RNA interacting amino acids. Next, we explored the genetic variability of the three qRRMs of human GRSF1 by searching genomic databases and tested the functional consequences of naturally occurring mutants. For this purpose the RNA-binding capacity of wild-type and mutant recombinant GRSF1 protein species was assessed by quantitative RNA electrophoretic mobility shift assays. We found that some of the naturally occurring GRSF1 mutants exhibited a strongly reduced RNA-binding activity although the general protein structure was hardly affected. These data suggested that homozygous allele carriers of these particular mutants express dysfunctional GRSF1 and thus may show defective GRSF1 signaling.     

Hematologic characterization of naturally occurring malaria (Plasmodium inui) in cynomolgus monkeys (Macaca fascicularis)

Twenty of 47 recently imported cynomolgus monkeys (Macaca fascicularis) were found to have malarial infections. The agent identified was Plasmodium inui. All infections were subclinical in nature. Parasitemias ranged from 10 to 900 parasites/mm3 of whole blood. Pre- and post-treatment hematologic values were evaluated following treatment with chloroquine. Treatment was effective in clearing parasitemias from 13 of 14 infected monkeys. Pretreatment values of hematocrit, hemoglobin, and mean corpuscular volume were significantly different in infected animals compared to noninfected animals. While post-treatment hemoglobin and hematocrit values returned to noninfected control levels, mean corpuscular volume values of infected animals remained significantly lower in the post-treatment period.     

The characterisation and identification of a naturally occurring hybrid in the genusLeucaena (Leguminosae: Mimosoideae)

Phytogeographical, morphological, and molecular evidence for the widespread but sporadic occurrence of sterile hybrids betweenLeucaena leucocephala subsp.glabrata andL. esculenta subsp.esculenta in South-Central Mexico is presented. Most morphological and DNA characters studied in the putative hybrids showed states intermediate between the proposed parental taxa. The occurrence of non-additive nuclear ribosomal DNA phenotypes is discussed and the need to use a suite of nuclear taxon-specific markers to determine hybridity is emphasized. The origin of the hybrid is discussed in relation to the disruption of the distributions of both parental taxa through use by man as minor food plants, providing another example of the important influence of human interference on the evolution ofLeucaena. The successful use of dried leaf material as a source of DNA is highlighted as an efficient way to identify sterile hybrids at the molecular level.     

Discovery of sexual dimorphisms in metabolic and genetic biomarkers

Metabolomic profiling and the integration of whole-genome genetic association data has proven to be a powerful tool to comprehensively explore gene regulatory networks and to investigate the effects of genetic variation at the molecular level. Serum metabolite concentrations allow a direct readout of biological processes, and association of specific metabolomic signatures with complex diseases such as Alzheimer's disease and cardiovascular and metabolic disorders has been shown. There are well-known correlations between sex and the incidence, prevalence, age of onset, symptoms, and severity of a disease, as well as the reaction to drugs. However, most of the studies published so far did not consider the role of sexual dimorphism and did not analyse their data stratified by gender. This study investigated sex-specific differences of serum metabolite concentrations and their underlying genetic determination. For discovery and replication we used more than 3,300 independent individuals from KORA F3 and F4 with metabolite measurements of 131 metabolites, including amino acids, phosphatidylcholines, sphingomyelins, acylcarnitines, and C6-sugars. A linear regression approach revealed significant concentration differences between males and females for 102 out of 131 metabolites (p-values<3.8×10(-4); Bonferroni-corrected threshold). Sex-specific genome-wide association studies (GWAS) showed genome-wide significant differences in beta-estimates for SNPs in the CPS1 locus (carbamoyl-phosphate synthase 1, significance level: p<3.8×10(-10); Bonferroni-corrected threshold) for glycine. We showed that the metabolite profiles of males and females are significantly different and, furthermore, that specific genetic variants in metabolism-related genes depict sexual dimorphism. Our study provides new important insights into sex-specific differences of cell regulatory processes and underscores that studies should consider sex-specific effects in design and interpretation.     

A naturally occurring genetic variant of human XRCC2 (R188H) confers increased resistance to cisplatin-induced DNA damage

Homologous recombination, a major double strand break repair pathway, plays critical roles in maintaining genome stability. Genetic polymorphisms in HR genes have been implicated in cancer risk. We report a novel assay system for evaluating polymorphisms in human homologous recombination genes using a panel of chicken DT40 repair mutants. We established mutant cell lines complemented with either wild-type or variant cDNAs of three human genes, RAD51, XRCC2, and XRCC3, and assessed their sensitivity to cisplatin and mitomycin C. DT40 mutants complemented with RAD51 coding and 5'UTR variants, and with a XRCC3 coding variant showed equivalent sensitivity as those with wild-type cDNAs. Interestingly, Xrcc2(-/-) DT40 cells complemented with variant XRCC2 (R188H) were more tolerant to cisplatin than those with wild-type XRCC2. Considering that the XRCC2 (R188H) allele reduces risk to epithelial ovarian cancer, the increased XRCC2 activity with the R188H polymorphism may have clinical benefit in preventing cancer risk.     

The pollination ecology of Actaea spicata (Ranunculaceae)

Pollination ecology of Actaea spicata L. (Ranunculaceae) was studied at localities ranging from deciduous to spruce forest. The species is self–incompatible and possibly facultatively apomictic. No anemogamy seems to occur. In deciduous forest, the plant is primarily pollinated by pollen–foraging and mate–searching Byturus ochraceus (Scriba) (Coleoptera). It flowers during a period between emergence of the beetle and onset of anthesis of the prime pollen source and only larval host plant, Geum ur–banum L. (Rosaceae). The beetles sit on the suitably long anther filaments or on top of the stigma. Floral morphology promotes pollination–performing movements by the beetle. The complex floral fragrance seems to induce close–range behaviour, i.e. alighting and/or foraging. Flies become abundant visitors only shortly before anthesis is over, and their contribution is small. Large syrphids are relatively effective as pollinators, since they grasp the stamens near the base and often touch the stigma with pollen–dusted parts of their bodies. Due to lack of its food–plant, B. ochraceus is absent at spruce forest localities and northwards. At these localities flowering is delayed until the main fly appearance, and the flowers are heavily pollinated by several syrphids and muscids. At three deciduous–forest localities a second flowering took place with new, juxtaposed inflorescences on separate stalks. The distribution of such inflorescences within the population along with the distribution and abundance of pollinators among the two generations of flowers suggest that these inflorescences occur as a response to low rate of pollination in the first anthesis.