Unravelling below-ground plant distributions: a real-time polymerase chain reaction method for quantifying species proportions in mixed root samples.

Knowledge on below-ground plant distributions is almost lacking to date, despite the fact that such information would be very valuable in understanding below-ground competition and species-specific interactions, processes that are expected to shape community structure. Methods available so far for below-ground species determination have drawbacks that we tried to challenge. Some methods make use of differences in the chemical composition between species, but this is highly variable upon environmental factors. DNA-based techniques - far less dependent on chemical composition - such as polymerase chain reaction on internal transcribed spacer (ITS) primers can so far only determine presence-absence of a species in a mixed root sample. Here, we present a quantitative DNA-based technique that allows investigation of relative species abundances in experimental mixed root samples. We used quantitative real-time polymerase chain reaction (PCR) on species-specific markers obtained from intersimple sequence repeat (ISSR) analyses in root samples. This molecular technique is novel in the field of root ecology and its development overcame three challenges: (i) determination of species-specific DNA fragments, (ii) development and optimization of the real time PCR protocol, (iii) designing a data treatment method based on a modified delta-delta-cycle threshold (CT) analysis. The method gained robustness from using relative DNA abundances in species mixtures rather than absolute concentration readings. This requires accurate multispecies reference series as a calibration. Test samples with different known biomass ratios of all species showed proof of concept of this method. The pro's and contra's of this method are discussed in the light of its contribution to advancing ecological research on below-ground plant-plant interactions.

Selection on phenotypic plasticity of morphological traits in response to flooding and competition in the clonal shore plant Ranunculus reptans.

Adaptive evolution of phenotypic plasticity requires that plastic genotypes have the highest global fitness. We studied selection by spatial heterogeneity of interspecific competition and flooding, and by temporal heterogeneity of flooding on morphological plasticity of 52 genotypes of the clonal shore plant Ranunculus reptans. Competition reduced clone size, rosette size, leaf length and stolon internode thickness. Flooding had similar effects and reduced competition. Differences in selection between environments imply potential for either local adaptation or for indirect evolution of phenotypic plasticity. We also detected direct selection for plastic reductions in internode length in response to flooding and for a plastic increase in internode length in response to competition. Plastic responses of some morphological traits to flooding were in line with selection thereon, suggesting that they indeed are adaptive and might have evolved in response to direct selection on plasticity.

Differential responses of germination and seedling establishment in populations of Tragopogon pratensis (Asteraceae).

The establishment phase is an important bottleneck in the life cycle of plants. It consists of two steps that are rarely separated, i.e., the germination of seeds and the establishment of seedlings. Here we report the results of two experiments in which we independently investigated germination and seedling establishment in the greenhouse, under different grass vegetation treatments representing different regeneration niches. Seeds of Tragopogon pratensis from six populations and two habitat types were studied, three from roadside verges and three from hayfields. Germination percentages and germination speed were higher for seeds from roadside verges than for seeds from hayfields, but were little affected by treatment. In contrast, seedling growth was much lower in the tall grass vegetation, than in the short grass and especially the bare soil treatment. Seedling sizes were generally similar for different populations and habitat types. Our results thus show that the two early steps in the establishment phase of plants may respond very differently to the micro-environment, and may have a different selection history. Insight into the ecology and evolution of life histories may require that germination and establishment are considered separately.

Limited costs of wrong root placement in Rumex palustris in heterogeneous soils.

Nutrient hot spots in the soil have a limited life span, but the costs and benefits for root foraging are still underexposed. We assessed short-term costs that may arise when a nutrient-rich patch induces root proliferation, but then rapidly disappears. Rumex palustris plants were grown with a homogeneous or a heterogeneous nutrient application. After root proliferation in a nutrient-rich patch, nutrient supply was switched from homogeneous to heterogeneous, and vice versa, or the patch location was changed. R. palustris proliferated its roots in the rich patch. After switching, the relative growth rates of the roots were adjusted to the novel pattern of nutrient availability. However, the changes in local root biomass lagged behind the rapid shift in nutrient supply, because the root mass realized in specific sectors could not be rapidly relocated. Despite this, R. palustris did not exhibit costs of switching in terms of biomass or nitrogen uptake. Our data suggest that rapid shifts in uptake rate and redistribution of nitrogen within the plant may have lowered the costs of incorrect root placement.

Density-induced plant size reduction and size inequalities in ethylene-sensing and ethylene-insensitive tobacco.

Plant competition for light is a commonly occurring phenomenon in natural and agricultural vegetations. It is typically size-asymmetric, meaning that slightly larger individuals receive a disproportionate share of the light, leaving a limited amount of light for the initially smaller individuals. As a result, size inequalities of such stands increase with competition intensity. A plant's ability to respond morphologically to the presence of neighbour plants with enhanced shoot elongation, the so-called shade avoidance response, acts against the development of size inequalities. This has been shown experimentally with transgenic plants that cannot sense neighbours and, therefore, show no shade avoidance responses. Stands of such transgenic plants showed a much stronger development of size inequalities at high plant densities than did wild type (WT) stands. However, the transgenic plants used in these experiments displayed severely hampered growth rates and virtually no response to neighbours. In order to more precisely study the impact of this phenotypic plasticity on size inequality development, experiments required plants that have normal growth rates and reduced, but not absent, shade avoidance responses. We made use of an ethylene-insensitive, transgenic tobacco genotype (Tetr) that has wild type growth rates and moderately reduced shade avoidance responses to neighbours. Here, we show that the development of size inequalities in monocultures of these plants is not affected unambiguously different from wild type monocultures. Plots of Tetr plants developed higher inequalities for stem length than did WT, but monocultures of the two genotypes had identical CV (Coefficient of Variance) values for shoot biomass that increased with plant density. Therefore, even though reduced shade avoidance capacities led to the expected higher size inequalities for stem length, this does not necessarily lead to increased size inequalities for shoot biomass.