Comparative population genetic structure in a plant-pollinator/seed predator system.

Comparative analyses of spatial genetic structure of populations of plants and the insects they interact with provide an indication of how gene flow, natural selection and genetic drift may jointly influence the distribution of genetic variation and potential for local co-adaptation for interacting species. Here, we analysed the spatial scale of genetic structure within and among nine populations of an interacting species pair, the white campion Silene latifolia and the moth Hadena bicruris, along a latitudinal gradient across Northern/Central Europe. This dioecious, short-lived perennial plant inhabits patchy, often disturbed environments. The moth H. bicruris acts both as its pollinator and specialist seed predator that reproduces by laying eggs in S. latifolia flowers. We used nine microsatellite markers for S. latifolia and eight newly developed markers for H. bicruris. We found high levels of inbreeding in most populations of both plant and pollinator/seed predator. Among populations, significant genetic structure was observed for S. latifolia but not for its pollinator/seed predator, suggesting that despite migration among populations of H. bicruris, pollen is not, or only rarely, carried over between populations, thus maintaining genetic structure among plant populations. There was a weak positive correlation between genetic distances of S. latifolia and H. bicruris. These results indicate that while significant structure of S. latifolia populations creates the potential for differentiation at traits relevant for the interaction with the pollinator/seed predator, substantial gene flow in H. bicruris may counteract this process in at least some populations.

EMS mutagenesis in mature seed-derived rice calli as a new method for rapidly obtaining tilling mutant populations.

Background: TILLING (Targeting Induced Local Lesions IN Genomes) is a reverse genetic method that combines chemical mutagenesis with high-throughput genome-wide screening for point mutation detection in genes of interest. However, this mutation discovery approach faces a particular problem which is how to obtain a mutant population with a sufficiently high mutation density. Furthermore, plant mutagenesis protocols require two successive generations (M1, M2) for mutation fixation to occur before the analysis of the genotype can begin. Results: Here, we describe a new TILLING approach for rice based on ethyl methanesulfonate (EMS) mutagenesis of mature seed-derived calli and direct screening of in vitro regenerated plants. A high mutagenesis rate was obtained (i.e. one mutation in every 451 Kb) when plants were screened for two senescence-related genes. Screening was carried out in 2400 individuals from a mutant population of 6912. Seven sense change mutations out of 15 point mutations were identified. Conclusions: This new strategy represents a significant advantage in terms of time-savings (i.e. more than eight months), greenhouse space and work during the generation of mutant plant populations. Furthermore, this effective chemical mutagenesis protocol ensures high mutagenesis rates thereby saving in waste removal costs and the total amount of mutagen needed thanks to the mutagenesis volume reduction.

Evolutionary Ecology of Mountain Birch in Subarctic Stress Gradients: Interplay of Biotic and Abiotic Factors in Plant-Plant Interactions and Evolutionary Processes

In nature, variation for example in herbivory, wind exposure, moisture and pollution impact often creates variation in physiological stress and plant productivity. This variation is seldom clear-cut, but rather results in clines of decreasing growth and productivity towards the high-stress end. These clines of unidirectionally changing stress are generally known as ‘stress gradients’. Through its effect on plant performance, stress has the capacity to fundamentally alter the ecological relationships between individuals, and through variation in survival and reproduction it also causes evolutionary change, i.e. local adaptations to stress and eventually speciation. In certain conditions local adaptations to environmental stress have been documented in a matter of just a few generations. In plant-plant interactions, intensities of both negative interactions (competition) and positive ones (facilitation) are expected to vary along stress gradients. The stress-gradient hypothesis (SGH) suggests that net facilitation will be strongest in conditions of high biotic and abiotic stress, while a more recent ‘humpback’ model predicts strongest net facilitation at intermediate levels of stress. Plant interactions on stress gradients, however, are affected by a multitude of confounding factors, making studies of facilitation-related theories challenging. Among these factors are plant ontogeny, spatial scale, and local adaptation to stress. The last of these has very rarely been included in facilitation studies, despite the potential co-occurrence of local adaptations and changes in net facilitation in stress gradients. Current theory would predict both competitive effects and facilitative responses to be weakest in populations locally adapted to withstand high abiotic stress. This thesis is based on six experiments, conducted both in greenhouses and in the field in Russia, Norway and Finland, with mountain birch (Betula pubescens subsp. czerepanovii) as the model species. The aims were to study potential local adaptations in multiple stress gradients (both natural and anthropogenic), changes in plant-plant interactions under conditions of varying stress (as predicted by SGH), potential mechanisms behind intraspecific facilitation, and factors confounding plant-plant facilitation, such as spatiotemporal, ontogenetic, and genetic differences. I found rapid evolutionary adaptations (occurring within a time-span of 60 to 70 years) towards heavy-metal resistance around two copper-nickel smelters, a phenomenon that has resulted in a trade-off of decreased performance in pristine conditions. Heavy-metal-adapted individuals had lowered nickel uptake, indicating a possible mechanism behind the detected resistance. Seedlings adapted to heavy-metal toxicity were not co-resistant to others forms of abiotic stress, but showed co-resistance to biotic stress by being consumed to a lesser extent by insect herbivores. Conversely, populations from conditions of high natural stress (wind, drought etc.) showed no local adaptations, despite much longer evolutionary time scales. Due to decreasing emissions, I was unable to test SGH in the pollution gradients. In natural stress gradients, however, plant performance was in accordance with SGH, with the strongest host-seedling facilitation found at the high-stress sites in two different stress gradients. Factors confounding this pattern included (1) plant size / ontogenetic status, with seedling-seedling interactions being competition dominated and host-seedling interactions potentially switching towards competition with seedling growth, and (2) spatial distance, with competition dominating at very short planting distances, and facilitation being strongest at a distance of circa ¼ benefactor height. I found no evidence for changes in facilitation with respect to the evolutionary histories of plant populations. Despite the support for SGH, it may be that the ‘humpback’ model is more relevant when the main stressor is resource-related, while what I studied were the effects of ‘non-resource’ stressors (i.e. heavy-metal pollution and wind). The results have potential practical applications: the utilisation of locally adapted seedlings and plant facilitation may increase the success of future restoration efforts in industrial barrens as well as in other wind-exposed sites. The findings also have implications with regard to the effects of global change in subarctic environments: the documented potential by mountain birch for rapid evolutionary change, together with the general lack of evolutionary ‘dead ends’, due to not (over)specialising to current natural conditions, increase the chances of this crucial forest-forming tree persisting even under the anticipated climate change.

Invasive populations of Senecio pterophorus are neither more productive nor more plastic than the native populations

Successful plant invaders may have specific morphological and physiological traits that promote invasion in a new habitat. The Evolution of Increased Competitive Ability (EICA) hypothesis predicts that plants released from natural enemies in the introduced habitats are more competitive and perform better than plants from the native populations. An increased phenotypic plasticity may also favour invasion because it allows plants to function under a wider range of environments. In this study we used Senecio pterophorus (Asteraceae) to test whether introduced plant populations are 1)more competitive and 2) more plastic compared with the native populations. We conducted a common garden experiment using plants from the native range (South Africa, Eastern Cape), an expanded range (South Africa, Western Cape) and two introduced ranges (Australia and Europe) under different conditions of water availability. Contrary to the EICA and the increased plasticity hypotheses, plants from the invasive and expanded populations grew less and responded less to watering than those from their native range. These results may be caused by a depleted competition as well as the presence of genetic bottlenecks in the newly invaded areas.

Detecting local adaptation in widespread grassland species - the importance of scale and local plant community

1 Adaptation of plant populations to local environments has been shown in many species but local adaptation is not always apparent and spatial scales of differentiation are not well known. In a reciprocal transplant experiment we tested whether: (i) three widespread grassland species are locally adapted at a European scale; (ii) detection of local adaptation depends on competition with the local plant community; and (iii) local differentiation between neighbouring populations from contrasting habitats can be stronger than differentiation at a European scale. 2 Seeds of Holcus lanatus, Lotus corniculatus and Plantago lanceolata from a Swiss, Czech and UK population were sown in a reciprocal transplant experiment at fields that exhibit environmental conditions similar to the source sites. Seedling emergence, survival, growth and reproduction were recorded for two consecutive years. 3 The effect of competition was tested by comparing individuals in weeded monocultures with plants sown together with species from the local grassland community. To compare large-scale vs. small-scale differentiation, a neighbouring population from a contrasting habitat (wet-dry contrast) was compared with the 'home' and 'foreign' populations. 4 In P. lanceolata and H. lanatus, a significant home-site advantage was detected in fitness-related traits, thus indicating local adaptation. In L. corniculatus, an overall superiority of one provenance was found. 5 The detection of local adaptation depended on competition with the local plant community. In the absence of competition the home-site advantage was underestimated in P. lanceolata and overestimated in H. lanatus. 6 A significant population differentiation between contrasting local habitats was found. In some traits, this small-scale was greater than large-scale differentiation between countries. 7 Our results indicate that local adaptation in real plant communities cannot necessarily be predicted from plants grown in weeded monocultures and that tests on the relationship between fitness and geographical distance have to account for habitat-dependent small-scale differentiation. Considering the strong small-scale differentiation, a local provenance from a different habitat may not be the best choice in ecological restoration if distant populations from a more similar habitat are available.

Genetic variability in natural populations of Zeyheria montana mart. from the Brazilian Cerrado

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitrogen redistribution to sorghum grains as affected by plant competition

An experiment was conducted to study nitrogen absorption and translocation in grain sorghum plants during their reproductive growth. Sorghum was grown in four row spacings: 50 and 70 cm in single rows, 80 and 120 cm in double rows 20 cm apart. Plant populations were 71000, 142000 and 213000 plants/ha. After flowering, samples were taken at 12 day intervals, and the plants were divided into grains and stover, where N was analyzed. There was an increase in N concentration in lower plant populations and in wider row spacings. However, total nitrogen accumulation (in kg/ha) increased as the number of plants was increased. In the vegetative parts of the plants there were higher N concentrations in lower populations showing that there was a higher N absorption and a lower translocation to the grains. When grain sorghum was grown in 50 cm rows, there was a high N accumulation, a high N translocation to the grains and the highest yield. This row spacing led to the highest N use efficiency.

Yield and quality of carrot seeds, cv. Brasilia, as a result of plant population, gibberellic acid and stage of maturity

'Brasilia' is a cultivar of carrot with characteristics suiting cultivation under hot conditions but with problems in seed production, arising from the conflicting requirements of root and seed production. One solution is to select cultivars requiring vernalisation and then to use GA(3) to induce flowering where the climate prevents this. There is, however, little information on plant population, seed maturity and harvesting time on which to base such a procedure. Accordingly this research was carried out to study the physiological quality and production of the seeds in plant populations from 25,000 to 800,000 plants/ha, in the seed-to-seed method of cv. Brasilia in Anapolis, GO, Brazil. In each population, two harvest methods (from first and second orders of umbels, or selected harvest, and remaining orders, or total harvest) and two stages of maturity (brownish, or mature seeds, and yellowish, or immature ones) were also evaluated. Two trials were carried out, with and without gibberellic acid. Seed was evaluated for physical characters, germination, vigour, 1000-seed weight, water content, dry matter and productivity. Seed was produced in both experiments (with or without GA3 spraying). Mature seed showed germination at least 30% greater than immature, and seed from the selected harvest showed germination 16% greater than from the total harvest. In increasing plant population to 200,000 plants/ha, seed quality was not affected, but productivity increased.

Peanut mechanized digging regarding to plant population and soil water level

The largest losses in mechanical harvesting of peanuts occur during the stage of digging, and its assessment is still incipient in Brazil. Therefore, the aim of this study was to evaluate the quantitative losses and the performance of the tractor-digger-inverter, according to soil water content and plant populations. The experiment was conducted in a completely randomized block design with a factorial scheme 2 x 3, in which the treatments consisted of two soil, water content (19.3 and 24.8%) and three populations of plants (86,111, 127,603 and 141,144 plants ha-1), with four replications. The quantitative digging losses and the set mechanized performance were evaluated. The largest amount of visible and total losses was found in the population of 141.144 plants ha-1 for the 19.3% soil water content. The harvested material flow and the tractor-digger-inverter performance were not influenced by soil water content and plant population. The water content in the pods was higher in 24.8% soil water content only for the population of 86,111 plants ha-1; the yield was higher in the populations of 141.144 and 127.603 plants ha-1, in the 19.3 e 24.8% soil water content, respectively.

ASIAN SOYBEAN RUST INTEGRATED MANAGEMENT POTENTIAL: DISEASE SEVERITY, PLANT DEVELOPMENT AND YIELD COMPONENTS, CULTIVAR MG/BR-46 (CONQUISTA)

Soybean rust caused by Phakopsora pachyrhizi Sydow & P. Sydow is one of the major diseases of the soybean crop. The aim of this study was to evaluate the effects of sowing dates, plant populations and reduced doses of fungicides on soybean rust severity and its effects on plant development and yield, cultivar MG/BR46 (Conquista). Field experiments were conducted in the 2009/2010 and 2010/2011 harvests, under natural rust infestation of soybean rust. As from the appearance of the first disease symptoms, also began the fungicide spraying and the disease severity assessments. To understand the nature and extent of the effects of different treatments, a multivariate analysis of factors was applied. For the majority of the agronomic characters and factors, one-third to two-thirds of their variability can be explained by changes in plant populations or by differences in the fungicide treatments, and the remainder, was explained by sowing date variations. The fungicide treatments and sowing dates are determinants in disease severity and its interference on crop productivity. The characters of plant growth are more dependent on plant population variations. Treatments with azoxystrobina + ciproconazol promoted smaller disease severities, reflecting in productivity increase. The plant populations can be reduced up to 160.000 plants ha(-1) without losses in the disease control and the soybean yield. In general, the earliest sowings provided increase in the plant development, although the rust control was less efficient.

Mating system parameters at hierarchical levels of fruits, individuals and populations in the Brazilian insect-pollinated tropical tree, Tabebuia roseo-alba (Bignoniaceae)

For many tree species, mating system analyses have indicated potential variations in the selfing rate and paternity correlation among fruits within individuals, among individuals within populations, among populations, and from one flowering event to another. In this study, we used eight microsatellite markers to investigate mating systems at two hierarchical levels (fruits within individuals and individuals within populations) for the insect pollinated Neotropical tree Tabebuia roseo-alba. We found that T. roseo-alba has a mixed mating system with predominantly outcrossed mating. The outcrossing rates at the population level were similar across two T. roseo-alba populations; however, the rates varied considerably among individuals within populations. The correlated paternity results at different hierarchical levels showed that there is a high probability of shared paternal parentage when comparing seeds within fruits and among fruits within plants and full-sibs occur in much higher proportion within fruits than among fruits. Significant levels of fixation index were found in both populations and biparental inbreeding is believed to be the main cause of the observed inbreeding. The number of pollen donors contributing to mating was low. Furthermore, open-pollinated seeds varied according to relatedness, including half-sibs, full-sibs, self-sibs and self- half-sibs. In both populations, the effective population size within a family (seed-tree and its offspring) was lower than expected for panmictic populations. Thus, seeds for ex situ conservation genetics, progeny tests and reforestation must be collected from a large number of seed-trees to guarantee an adequate effective population in the sample.

Comparison of microsatellites and isozymes in genetic diversity studies of Oryza glumaepatula (Poaceae) populations

The study of the genetic structure of wild plant populations is essential for their management and conservation. Several DNA markers have been used in such studies, as well as isozyme markers. In order to provide a better comprehension of the results obtained and a comparison between markers which will help choose tools for future studies in natural populations of Oryza glumaepatula, a predominantly autogamous species, this study used both isozymes and microsatellites to assess the genetic diversity and genetic structure of 13 populations, pointing to similarities and divergences of each marker, and evaluating the relative importance of the results for studies of population genetics and conservation. A bulk sample for each population was obtained, by sampling two to three seeds of each plant, up to a set of 50 seeds. Amplified products of eight SSR loci were electrophoresed on non-denaturing polyacrylamide gels, and the fragments were visualized using silver staining procedure. Isozyme analyses were conducted in polyacrylamide gels, under a discontinuous system, using six enzymatic loci. SSR loci showed higher mean levels of genetic diversity (A=2.83, p=0.71, A(P)=3.17, H-o=0.081, H-e=0.351) than isozyme loci (A=1.20, p=0.20, A(P)=1.38, H-o=0.006, H-e=0.056). Interpopulation genetic differentiation detected by SSR loci (R-ST=0.631, equivalent to F-ST=0.533) was lower than that obtained with isozymes (F-ST=0.772). However, both markers showed high deviation from Hardy-Weinberg expectations (F-IS=0.744 and 0.899, respectively for SSR and isozymes). The mean apparent outcrossing rate for SSR ((t) over bar (a)=0.14) was higher than that obtained using isozymes ((t) over bar (a)=0.043), although both markers detected lower levels of outcrossing in Amazonia compared to the Pantanal. The migrant number estimation was also higher for SSR (Nm=0.219) than isozymes (Nm=0.074), although a small number for both markers was expected due to the mode of reproduction of this species, defined as mixed with predominance of self fertilization. No correlation was obtained between genetic and geographic distances with SSR, but a positive correlation was found between genetic and geographic distances with isozymes. We conclude that these markers are divergent in detecting genetic diversity parameters in O. glumaepatula and that microsatellites are powerful for detecting information at the intra-population level, while isozymes are more powerful for inter-population diversity, since clustering of populations agreed with the expectations based on the geographic distribution of the populations using this marker. Rev. Biol. Trop. 60 (4): 1463-1478. Epub 2012 December 01.

Consequences of plant population size for pollinator visitation and plant reproductive success

Habitat loss and fragmentation have a prominent role in determining the size of plant populations, and can affect plant-pollinator interactions. It is hypothesized that in small plant populations the ability to set seeds can be reduced due to limited pollination services, since individuals in small populations can receive less quantity or quality of visits. In this study, I investigated the effect of population size on plant reproductive success and insect visitation in 8 populations of two common species in the island of Lesvos, Greece (Mediterranean Sea), Echium plantagineum and Ballota acetabulosa, and of a rare perennial shrub endemic to north-central Italy, Ononis masquillierii. All the three species depended on insect pollinators for sexual reproduction. For each species, pollen limitation was present in all or nearly all populations, but the relationship between pollen limitation and population size was only present in Ononis masquillierii. However, in Echium plantagineum, significant relationships between both open-pollinated and handcrossed-pollinated seed sets and population size were found, being small populations comparatively less productive than large ones. Additionally, for this species, livestock grazing intensity was greater for small populations and for sparse patches, and had a negative influence on productivity of the remnant plants. Both Echium plantagineum and Ballota acetabulosa attracted a great number of insects, representing a wide spectrum of pollinators, thereby can be considered as generalist species. For Ballota acetabulosa, the most important pollinators were megachilid female bees, and insect diversity didn’t decrease with decreasing plant population size. By contrast, Ononis masquillierii plants generally received few visits, with flowers specialized on small bees (Lasioglossum spp.), representing the most important insect guild. In Echium plantagineum and Ballota acetabulosa, plants in small and large populations received the same amount of visits per flower, and no differences in the number of intraplant visited flowers were detected. On the contrary, large Ononis populations supported higher amounts of pollinators than small ones. At patch level, high Echium flower density was associated with more and higher quality pollinators. My results indicate that small populations were not subject to reduced pollination services than large ones in Echium plantagineum and Ballota acetabulosa, and suggest that grazing and resource limitation could have a major impact on population fitness in Echium plantagineum. The absence of any size effects in these two species can be explained in the light of their high local abundance, wide habitat specificity, and ability to compete with other co-flowering species for pollinators. By contrast, size represents a key characteristic for both pollination and reproduction in Ononis masquillierii populations, as an increase in size could mitigate the negative effects coming from the disadvantageous reproductive traits of the species. Finally, the widespread occurrence of pollen limitation in the three species may be the result of 1) an ongoing weakening or disruption of plantpollinator interactions derived from ecological perturbations, 2) an adaptive equilibrium in response to stochastic processes, and 3) the presence of unfavourable reproductive traits (for Ononis masquillierii).

Between-population outbreeding affects plant defence

Between-population crosses may replenish genetic variation of populations, but may also result in outbreeding depression. Apart from direct effects on plant fitness, these outbreeding effects can also alter plant-herbivore interactions by influencing plant tolerance and resistance to herbivory. We investigated effects of experimental within- and between-population outbreeding on herbivore resistance, tolerance and plant fitness using plants from 13 to 19 Lychnis flos-cuculi populations. We found no evidence for outbreeding depression in resistance reflected by the amount of leaf area consumed. However, herbivore performance was greater when fed on plants from between-population compared to within-population crosses. This can reflect outbreeding depression in resistance and/or outbreeding effects on plant quality for the herbivores. The effects of type of cross on the relationship between herbivore damage and plant fitness varied among populations. This demonstrates how between-population outbreeding effects on tolerance range from outbreeding depression to outbreeding benefits among plant populations. Finally, herbivore damage strengthened the observed outbreeding effects on plant fitness in several populations. These results raise novel considerations on the impact of outbreeding on the joint evolution of resistance and tolerance, and on the evolution of multiple defence strategies.

Effects of experimental inbreeding on herbivore resistance and plant fitness: the role of history of inbreeding, herbivory and abiotic factors

Inbreeding is common in plant populations and can affect plant fitness and resistance against herbivores. These effects are likely to depend on population history. In a greenhouse experiment with plants from 17 populations of Lychnis flos-cuculi, we studied the effects of experimental inbreeding on resistance and plant fitness. Depending on the levels of past herbivory and abiotic factors at the site of plant origin, we found either inbreeding or outbreeding depression in herbivore resistance. Furthermore, when not damaged experimentally by snail herbivores, plants from populations with higher heterozygosity suffered from inbreeding depression and those from populations with lower heterozygosity suffered from outbreeding depression. These effects of inbreeding and outbreeding were not apparent under experimental snail herbivory. We conclude that inbreeding effects on resistance and plant fitness depend on population history. Moreover, herbivory can mask inbreeding effects on plant fitness. Thus, understanding inbreeding effects on plant fitness requires studying multiple populations and considering population history and biotic interactions.