The role of climatic tolerances and seed traits in reduced extinction rates of temperate polygonaceae.

The latitudinal diversity gradient (LDG) is one of the most striking and consistent biodiversity patterns across taxonomic groups. We investigate the species richness gradient in the buckwheat family, Polygonaceae, which exhibits a reverse LDG and is, thus, decoupled from dominant gradients of energy and environmental stability that increase toward the tropics and confound mechanistic interpretations. We test competing age and evolutionary diversification hypotheses, which may explain the diversification of this plant family over the past 70 million years. Our analyses show that the age hypothesis, which posits that clade richness is positively correlated with the ecological and evolutionary time since clade origin, fails to explain the richness gradient observed in Polygonaceae. However, an evolutionary diversification hypothesis is highly supported, with diversification rates being 3.5 times higher in temperate clades compared to tropical clades. We demonstrate that differences in rates of speciation, migration, and molecular evolution insufficiently explain the observed patterns of differential diversification rates. We suggest that reduced extinction rates in temperate clades may be associated with adaptive responses to selection, through which seed morphology and climatic tolerances potentially act to minimize risk in temporally variable environments. Further study is needed to understand causal pathways among these traits and factors correlated with latitude.

Seed-specific silencing of OsMRP5 reduces seed phytic acid and weight in rice.

Phytic acid (PA) is poorly digested by humans and monogastric animals and negatively affects human/animal nutrition and the environment. Rice mutants with reduced PA content have been developed but are often associated with reduced seed weight and viability, lacking breeding value. In the present study, a new approach was explored to reduce seed PA while attaining competitive yield. The OsMRP5 gene, of which mutations are known to reduce seed PA as well as seed yield and viability, was down-regulated specifically in rice seeds by using an artificial microRNA driven by the rice seed specific promoter Ole18. Seed PA contents were reduced by 35.8-71.9% in brown rice grains of transgenic plants compared to their respective null plants (non-transgenic plants derived from the same event). No consistent significant differences of plant height or number of tillers per plant were observed, but significantly lower seed weights (up to 17.8% reduction) were detected in all transgenic lines compared to null plants, accompanied by reductions of seed germination and seedling emergence. It was observed that the silencing of the OsMRP5 gene increased the inorganic P (Pi) levels (up to 7.5 times) in amounts more than the reduction of PA-P in brown rice. This indicates a reduction in P content in other cellular compounds, such as lipids and nucleic acids, which may affect overall seed development. Put together, the present study demonstrated that seed specific silencing of OsMRP5 could significantly reduce the PA content and increase Pi levels in seeds; however, it also significantly lowers seed weight in rice. Discussions were made regarding future directions towards producing agronomically competitive and nutritionally valuable low PA rice.

Pre-dispersal predation effect on seed packaging strategies and seed viability.

An increased understanding of intraspecific seed packaging (i.e. seed size/number strategy) variation across different environments may improve current knowledge of the ecological forces that drive seed evolution in plants. In particular, pre-dispersal seed predation may influence seed packaging strategies, triggering a reduction of the resources allocated to undamaged seeds within the preyed fruits. Assessing plant reactions to pre-dispersal seed predation is crucial to a better understanding of predation effects, but the response of plants to arthropod attacks remains unexplored. We have assessed the effect of cone predation on the size and viability of undamaged seeds in populations of Juniperus thurifera with contrasting seed packaging strategies, namely, North African populations with single-large-seeded cones and South European populations with multi-small-seeded cones. Our results show that the incidence of predation was lower on the single-large-seeded African cones than on the multi-small-seeded European ones. Seeds from non-preyed cones were also larger and had a higher germination success than uneaten seeds from preyed cones, but only in populations with multi-seeded cones and in cones attacked by Trisetacus sp., suggesting a differential plastic response to predation. It is possible that pre-dispersal seed predation has been a strong selective pressure in European populations with high cone predation rates, being a process which maintains multi-small-seeded cones and empty seeds as a strategy to save some seeds from predation. Conversely, pre-dispersal predation might not have a strong effect in the African populations with single-large-seeded cones characterized by seed germination and filling rates higher than those in the European populations. Our results indicate that differences in pre-dispersal seed predators and predation levels may affect both selection on and intraspecific variation in seed packaging.

Production of low phytic acid rice by hairpin RNA- and artificial microRNA-mediated silencing of OsMIK in seeds

To produce agronomically competitive rice with nutritionally superior, environmentally safe phytic acid (PA) levels, hairpin RNA (hpRNA)- and artificial microRNA (amiRNA)-mediated gene silencing approaches were explored to reduce both myo-inositol kinase gene (OsMIK) expression and PA accumulation in rice seeds. hpRNA and amiRNA sequences targeted to OsMIK (hpMIK and amiMIK), under the control of a rice Ole18 promoter, were transformed into the rice cultivar Nippon-bare. Fourteen and 21 independent transgenic events were identified containing the hpMIK and amiMIK constructs, respectively, from which five stable homozygous transgenic lines of each were developed together with their null siblings. Southern blotting demonstrated transgene integration into the genome and quantitative real-time PCR showed that gene silencing was restricted to seeds. OsMIK transcripts were significantly reduced in both transgenic amiMIK and hpMIK seeds, which had PA levels reduced by 14.9-50.2 and 38.1-50.7 %, respectively, compared with their respective null siblings. There were no systematic significant differences in agronomic traits between the transgenic lines and their non-transgenic siblings, and no correlation between seed PA contents and decreased rates of seed germination and seedling emergence. The results of the present study suggest that Ole 18-driven OsMIK silencing via hpRNA and amiRNA could be an effective way to develop agronomically competitive low phytic acid rice.

DNA banking for plant breeding, biotechnology and biodiversity evaluation.

The manipulation of DNA is routine practice in botanical research and has made a huge impact on plant breeding, biotechnology and biodiversity evaluation. DNA is easy to extract from most plant tissues and can be stored for long periods in DNA banks. Curation methods are well developed for other botanical resources such as herbaria, seed banks and botanic gardens, but procedures for the establishment and maintenance of DNA banks have not been well documented. This paper reviews the curation of DNA banks for the characterisation and utilisation of biodiversity and provides guidelines for DNA bank management. It surveys existing DNA banks and outlines their operation. It includes a review of plant DNA collection, preservation, isolation, storage, database management and exchange procedures. We stress that DNA banks require full integration with existing collections such as botanic gardens, herbaria and seed banks, and information retrieval systems that link such facilities, bioinformatic resources and other DNA banks. They also require efficient and well-regulated sample exchange procedures. Only with appropriate curation will maximum utilisation of DNA collections be achieved.

Population differentiation and its consequences in the white campion "Silene latifolia" (Caryophyllaceae): a functional analysis

Summary In his theory On the Origin of Species by Means of Natural Selection (1859), Darwin describes evolution as a gradual change in population over time and that natural selection is a process that caused evolution. Because quantitative variation in species is partly influenced by several genes and thus heritable, association between levels of genetic variation at neutral markers and at quantitative traits and their partitioning within and among populations are important to study mechanisms that drive evolution in populations. Most studies addressing quantitative variation in plants focused on morphological and life history traits but not in traits affecting reproductive success. The aim of this thesis is to better understand how patterns of variation for neutral molecular markers and phenotypic traits drive the evolution of reproduction and defensive mechanisms in six European populations of Silene latifolia, a dioecious plant species. We found evidence for extremely high within and between population variation at six microsatellite loci and at most quantitative traits studied in plants grown under standardized conditions (morphology, life history and reproductive traits). Interestingly, there was clinal variation between age at first flowering and latitude. This pattern is likely due to natural selection since differentiation of this trait was high, heritable and probably higher than differentiation at neutral markers. Our study focused on sex specific selective pressures: mechanisms of intersexual coadaptation and defence mechanism against the seed predator Hadena bicruris. To address divergence at reproductive traits, we studied male and female population of origin effects and in particular pollen competitive ability on male post-pollination success in the study populations with within and between populations crosses. We crossed the same female plant with pollen from a male within the same population of origin and pollen from two males from two distinct populations, using a fixed tester male as a competitor. Additionally, we conducted control crosses with pollen from each male as a single donor. We analysed paternity success of each competitor with two microsatellite loci, seed set and offspring fitness. Male population of origin showed significant among-population variation for siring success at pollen competition. In vitro pollen germination rate showed heritable variation among populations and was positively correlated to siring success. Local or foreign pollen did not have a consistent advantage. Furthermore, female population of origin affected the outcome of pollen competition in some populations. There was no difference of seed set or offspring fitness in within/ between population crosses. This suggests that reproductive divergence may occur via pollen competition in Silene latifolia. The specialist seed predator Hadena bicruris may also induce divergence between populations. We tested potential constitutive and induced defence mechanisms against the specialist predator Hadena bicruris. Because fruit wall thickness is smaller in the invasive range (Northern America) were the moth is absent, this suggests that a thicker fruit wall is a potentially defensive trait against larval attack, and that relaxed selection in the absence of the seed predator has resulted in an evolutionary loss of this defence in the invasive range. Fruit wall thickness was different among three populations. Experimental exposure to moth eggs increased fruit abortion. Fruits built after attack on exposed plants did not have thicker fruit walls compared to fruits on non-exposed plants. Furthermore, fruits with thicker fruit walls were not less profitable, nor did they require longer handling time when exposed to larvae, suggesting no defensive role of fruit wall thickness. Our results show that there is high molecular and phenotypic variation in Silene latifolia and that traits potentially involved in reproductive success both for intra-specific (between sexes) and inter-specific interactions are heritable. Different selective forces may thus interact and cause differential evolution of geographically separated Silene latifolia populations in Europe, leading to the observed differentiation. Résumé Dans sa théorie de l'évolution, L'origine des espèces, ch. 4 (1859), Darwin décrit l'évolution comme un processus continu au cours du temps à l'intérieur de populations et que la sélection naturelle en est le moteur. La variation quantitative est en partie déterminée par plusieurs gènes, donc transmissible à la descendance. Associer le niveau de variation génétique à des marqueurs neutres au niveau de la variation à des traits quantitatifs, ainsi que la répartition à l'intérieur et entre les populations d'une espèce donnée de cette variation, sont importants dans la compréhension des forces évolutives. La plupart des études scientifiques sur la variation quantitative chez les plantes se sont intéressées à la morphologie et à la phénologie mais pas aux caractères impliqués dans le succès reproducteur. L'objectif de cette thèse est de mieux comprendre comment la répartition de la variation à des marqueurs neutres et des caractères quantitatifs influence l'évolution de la reproduction et des mécanismes de défense dans six populations Européennes de l'espèce dioïque Silene latifolia. Nous avons mis en évidence une grande diversité intra et inter-population à six loci microsatellites ainsi qu'à la plupart des caractères quantitatifs mesurés (morphologie, phénologie et traits reproducteurs) sur des plantes cultivées dans des conditions standardisées. Un résultat intéressant est la présence d'un cline latitudinal pour l'âge à la floraison. Ceci est probablement une conséquence de la sélection naturelle, puisque ce caractère est différencié entre les populations étudiées, héritable et que la différenciation de ce trait est supérieure à la différenciation des marqueurs neutres étudiés. Notre étude a ensuite porté plus précisément sur les pressions de sélection spécifiques aux sexes : la coadaptation entre les sexes et les mécanismes de défense contre l'insecte granivore Hadena bicruris. Afin d'évaluer la divergence sur les traits reproducteurs, nous avons étudié les effets des populations d'origine des mâles et des femelles et en particulier le succès reproducteur des mâles après pollinisation à l'aide de croisements inter et intra-population. Nous avons pollinisé la même femelle avec du pollen provenant d'un mâle de la même population ainsi qu'avec le pollen de deux mâles provenant de deux autres populations en situation de compétition avec un pollen provenant d'une population test. Des croisements contrôle ont été réalisés avec les mêmes mâles en pollinisation pure. Nous avons évalué le succès reproducteur de chaque mâle à l'aide d'analyses de paternité ainsi que la production de graines et la fitness de la descendance. L'origine du mâle avait un effet sur la paternité. Le taux de croissance in vitro du pollen est un caractère héritable et a eu un effet positif sur le succès reproducteur. De plus, l'origine de la femelle avait un effet sur le succès des mâles en compétition dans certaines populations. Nos résultats suggèrent qu'une divergence reproductive chez Silene latifolia pourrait apparaître suite à la compétition pollinique. Nous avons ensuite testé des mécanismes potentiels de défense constitutive et induite contre l'herbivore spécialiste Hadena bicruris, un papillon nocturne qui pourrait aussi jouer un rôle dans la différenciation des populations. L'épaisseur des fruits étant plus faible dans les régions où la plante est invasive (Amérique du Nord) et où l'insecte est absent, ce trait pourrait jouer un rôle défensif. Une pression de sélection plus faible causée par l'absence de l'herbivore aurait abouti à une perte de cette défense dans ces régions. Nous avons montré que l'épaisseur du fruit est variable selon les populations. L'infestation artificielle de fruit par l'insecte induit l'abscission sélective des fruits. Les fruits produits après une infestation n'étaient pas plus épais que les fruits issus de plantes non infestées. De plus, les fruits épais n'étaient pas moins nutritifs et ne causaient pas de perte de temps pour la prédation pour les larves, ce qui suggère que l'épaisseur des fruits ne joue pas un rôle défensif. Nos résultats montrent que plusieurs pressions de sélection interviennent et interagissent dans l'évolution de populations distantes, provoquant la divergence des populations Européennes de l'espèce Silene latifolia.

MRI-based prostate brachytherapy seed localization.

A magnetic resonance imaging (MRI) pulse sequence and a corresponding image processing algorithm to localize prostate brachytherapy seeds during or after therapy are presented. Inversion-Recovery with ON-resonant water suppression (IRON) is an MRI methodology that generates positive contrast in regions of magnetic field susceptibility, as created by prostate brachytherapy seeds. Phantoms comprising of several materials found in brachytherapy seeds were created to assess the usability of the IRON pulse sequence for imaging seeds. Resulting images show that seed materials are clearly visible with high contrast using IRON, agreeing with theoretical predictions. A seed localization algorithm to process IRON images demonstrates the potential of this imaging technique for seed localization and dosimetry.

Predicting current and future spatial community patterns of plant functional traits

Community-level patterns of functional traits relate to community assembly and ecosystem functioning. By modelling the changes of different indices describing such patterns - trait means, extremes and diversity in communities - as a function of abiotic gradients, we could understand their drivers and build projections of the impact of global change on the functional components of biodiversity. We used five plant functional traits (vegetative height, specific leaf area, leaf dry matter content, leaf nitrogen content and seed mass) and non-woody vegetation plots to model several indices depicting community-level patterns of functional traits from a set of abiotic environmental variables (topographic, climatic and edaphic) over contrasting environmental conditions in a mountainous landscape. We performed a variation partitioning analysis to assess the relative importance of these variables for predicting patterns of functional traits in communities, and projected the best models under several climate change scenarios to examine future potential changes in vegetation functional properties. Not all indices of trait patterns within communities could be modelled with the same level of accuracy: the models for mean and extreme values of functional traits provided substantially better predictive accuracy than the models calibrated for diversity indices. Topographic and climatic factors were more important predictors of functional trait patterns within communities than edaphic predictors. Overall, model projections forecast an increase in mean vegetation height and in mean specific leaf area following climate warming. This trend was important at mid elevation particularly between 1000 and 2000 m asl. With this study we showed that topographic, climatic and edaphic variables can successfully model descriptors of community-level patterns of plant functional traits such as mean and extreme trait values. However, which factors determine the diversity of functional traits in plant communities remains unclear and requires more investigations.

Beta-oxidation in fatty acid degradation and beyond.

The degradation of fatty acids in plants occurs primarily in the peroxisomes through the beta-oxidation cycle. Enzymes that are involved in various aspects of beta-oxidation have been identified recently and shown to act biochemically on a diversity of fatty acids and derivatives. Analysis of several mutants has revealed essential roles for beta-oxidation in the breakdown of reserve triacylglycerols, seed development, seed germination and post-germinative growth before the establishment of photosynthesis. Beta-oxidation has also a considerable importance during the vegetative and reproductive growth phases, and plays a role in plant responses to stress, particularly in the synthesis of jasmonic acid.

Environment-dependent microevolution in a Mediterranean pine (Pinus pinaster Aiton).

BACKGROUND: A central question for understanding the evolutionary responses of plant species to rapidly changing environments is the assessment of their potential for short-term (in one or a few generations) genetic change. In our study, we consider the case of Pinus pinaster Aiton (maritime pine), a widespread Mediterranean tree, and (i) test, under different experimental conditions (growth chamber and semi-natural), whether higher recruitment in the wild from the most successful mothers is due to better performance of their offspring; and (ii) evaluate genetic change in quantitative traits across generations at two different life stages (mature trees and seedlings) that are known to be under strong selection pressure in forest trees. RESULTS: Genetic control was high for most traits (h2 = 0.137-0.876) under the milder conditions of the growth chamber, but only for ontogenetic change (0.276), total height (0.415) and survival (0.719) under the more stressful semi-natural conditions. Significant phenotypic selection gradients were found in mature trees for traits related to seed quality (germination rate and number of empty seeds). Moreover, female relative reproductive success was significantly correlated with offspring performance for specific leaf area (SLA) in the growth chamber experiment, and stem mass fraction (SMF) in the experiment under semi-natural conditions, two adaptive traits related to abiotic stress-response in pines. Selection gradients based on genetic covariance of seedling traits and responses to selection at this stage involved traits related to biomass allocation (SMF) and growth (as decomposed by a Gompertz model) or delayed ontogenetic change, depending also on the testing environment. CONCLUSIONS: Despite the evidence of microevolutionary change in adaptive traits in maritime pine, directional or disruptive changes are difficult to predict due to variable selection at different life stages and environments. At mature-tree stages, higher female effective reproductive success can be explained by differences in their production of offspring (due to seed quality) and, to a lesser extent, by seemingly better adapted seedlings. Selection gradients and responses to selection for seedlings also differed across experimental conditions. The distinct processes involved at the two life stages (mature trees or seedlings) together with environment-specific responses advice caution when predicting likely evolutionary responses to environmental change in Mediterranean forest trees.

Evidence for adaptive radiation from a phylogenetic study of plant defenses.

One signature of adaptive radiation is a high level of trait change early during the diversification process and a plateau toward the end of the radiation. Although the study of the tempo of evolution has historically been the domain of paleontologists, recently developed phylogenetic tools allow for the rigorous examination of trait evolution in a tremendous diversity of organisms. Enemy-driven adaptive radiation was a key prediction of Ehrlich and Raven's coevolutionary hypothesis [Ehrlich PR, Raven PH (1964) Evolution 18:586-608], yet has remained largely untested. Here we examine patterns of trait evolution in 51 North American milkweed species (Asclepias), using maximum likelihood methods. We study 7 traits of the milkweeds, ranging from seed size and foliar physiological traits to defense traits (cardenolides, latex, and trichomes) previously shown to impact herbivores, including the monarch butterfly. We compare the fit of simple random-walk models of trait evolution to models that incorporate stabilizing selection (Ornstein-Ulenbeck process), as well as time-varying rates of trait evolution. Early bursts of trait evolution were implicated for 2 traits, while stabilizing selection was implicated for several others. We further modeled the relationship between trait change and species diversification while allowing rates of trait evolution to vary during the radiation. Species-rich lineages underwent a proportionately greater decline in latex and cardenolides relative to species-poor lineages, and the rate of trait change was most rapid early in the radiation. An interpretation of this result is that reduced investment in defensive traits accelerated diversification, and disproportionately so, early in the adaptive radiation of milkweeds.