Variable herkogamy and autofertility in marginal populations ofGentianella germanica in the Netherlands

Gentianella germanica (Willd.) Börner, a rare plant species, occurs at the margin of its distribution area in the Netherlands. It is found only in isolated patches of chalk grassland surrounded by intensively-used agricultural land. In 1997 and 1998, we investigated the spatial and temporal separation of anthers and stigma in relation to autofertility. We performed our experiments in one large and two small populations to investigate the possible selection against herkogamy and for increased autofertility in marginal populations with pollination limitation. Dichogamy appeared to be very weak and varied within individuals. Homogamous and protandrous flowers were most frequent. Dichogamy is apparently no effective barrier against self-fertilization in this species. Herkogamy varied significantly between individuals and between populations, and plant and flower size were not correlated with anther-stigma separation. Both observations suggest a genetic basis for herkogamy. Autofertility was generally high in all three populations. Only in one small population was a correlation between autofertility and herkogamy found. In this population, most plants had the stigma positioned above the anthers (mean herkogamy +0.8 mm), while in the other two populations the stigma was positioned mainly in between or below the anthers (mean herkogamy ?0.55 mm). In comparison with earlier data from 1991 and 1992, plants in the large population were less herkogamous and more self-fertile in 1998. Since 1993 this population was mown each year one month earlier than usual, leading to dramatic reductions (bottlenecks) in population size. In addition, the early mowing time may have caused a further reduction of the already scarce visitation by pollinating insects. We hypothesize that both adverse conditions may have caused a selection of plants with smaller anther-stigma separation and higher autofertility and thus showing that human influence can have profound effects on the reproductive success and evolution of rare plant species, even if it occurs in the context of nature conservation.     

Hairpins involving both inverted and direct repeats are associated with homoplasious indels in non-coding chloroplast DNA of Taraxacum (Lactuceae: Asteraceae).

Sequence variation in 2.2 kb of non-coding regions of the chloroplast genome of eight dandelions (Taraxacum: Lactuceae) from Asia and Europe is interpreted in the light of the phylogenetic signal of base substitutions vs. indels (insertions-deletions). The four non-coding regions displayed a total of approximately 30 structural mutations of which 9 are potentially phylogenetically informative. Insertions, deletions, and an inversion were found that involved consecutive stretches of up to 172 bases. When compared to phylogenetic relationships of the chloroplast genomes based on nucleotide substitutions only, many homoplasious indels (33%) were detected that differed considerably in length and did not comprise simple sequence repeats typically associated with replication slippage. Though many indels in the intergenic spacers were associated with direct repeats, frequently, the variable stretches participated in inverted repeat stabilized hairpins. In each intergenic spacer or intron examined, nucleotide stretches ranging from 30 to 60 bp were able to fold into stabilized secondary structures. When these indels were homoplasious, they always ranked among the most stabilized hairpins in the non-coding regions. The association of higher order structures that involve both classes of repeats and parallel structural mutations in hot spot regions of the chloroplast genome can be used to differentiate among mutations that differ in phylogenetic reliability.     

Anthropogenic disturbance and habitat differentiation between sexual diploid and apomictic triploidTaraxacum sect.Ruderalia

Co-occurrence of sexual diploid and apomictic triploidTaraxacum sectionRuderalia has been reported frequently. Many suggestions have been put forward with respect to the existence of an ecological differentiation between the cytotypes. In a study of 116Ruderalia populations in the area around Neuchatel (Switzerland) such a habitat differentiation has been proven. Large differences in the diploid/triploid ratio have been found between different field types. Adjacent fields of different types sometimes had significantly different cytotype ratios. In a canonical correspondence analysis significant vegetation differences were found between fields with mainly diploidRuderalia and fields with mainly triploidRuderalia. Two environmental indicators were significantly positively correlated with ploidy levels, altitude with diploids, and the percentage of therophytes in a vegetation with triploids. The percentage of therophytes is thought to be an indicator of the amount of disturbance due to human activities. At lower altitudes, the diploid/triploid ratio is largely determined by the amount of disturbance. Highly-disturbed fields contain mainly triploids whereas relatively stable fields contain mainly diploids. At higher altitudes there are mainly diploids. The presence of triploidTaraxacum sect.Ruderalia in the region around Neuchatel appears to be the result of the high amount of human activities of the last few hundred years. Whether the triploid presence is due to immigration or to local formation from diploids is yet unknown.     

Biodiversity and agro-ecology in field margins

This multidisciplinary study investigates agro-ecological functions (nature conservation, agriculture, environment) and implications of newly created, mown sown and unsown field margin strips installed on ex-arable land to increase biodiversity. From conservational concern, the development of species rich field margin strips was not strongly affected by the installed type of margin strip since species diversity converged over time, whether strips were sown or not. Convergence between unsown and sown margin strips occurred also in terms of species composition: unsown and sown strips became similar over time. Mowing without removal of cuttings significantly reduced species richness, yielded more grassy margin strips and delayed similarity in species composition between sown and unsown margin strips. Species richness on the longer term was not significantly affected by light regime nor by disturbance despite significant temporary effects shortly after the disturbance event. On the contrary vegetation composition in terms of importance of functional groups changed after disturbance: the share of spontaneous species within functional groups increased resulting in higher similarity between the sown and unsown vegetation. Furthermore risk of invasion was highest in the disturbed unsown community on the unshaded side of a tree lane. A positive effect of botanical diversity on insect number and diversity was found. However the effects of botanical diversity on insect number was mediated by light regime. At high light availability differences between plant communities were more pronounced compared to low light availablilty. The abundance of some insect families was dependent on the vegetation composition. Furthermore light availability significantly influenced insect diversity as well as the spatial distribution of families. From agricultural concern, installing margin strips by sowing a species mixture and a mowing regime with removal of cuttings are good practices to diminish the risk of species ingrowth into adjacent crops by creeping roots and rhizomes. Seed dispersal was only problematic one year after the installation of the field margin strips particularly nearby the unsown margin strip and wind-borne seeds were dispersed over limited distances, mainly within 4 m of field margins. Annual herbage yield was not affected by mowing management. DM yield of sown/unsown communities converged over time. Compared to herbage from an intensively managed fertilized grassland, field margin herbage revealed a low feeding value, owing to a low crude protein content, a low digestibility and a high crude fibre content. The unsown community had a higher forage quality than communities sown to bred commercially available grass varieties. Both digestibility and crude protein content decreased over time irrespective of plant community or location. Mid June cuts were more productive than mid September cuts but digestibility and crude protein content was lower. The use of herbage from field margins as hay for horses or as a component in farmland compost are good alternatives. A strong relation was found between the distribution of pest insects and their antagonist families along field margin strips indicating a status of biological equilibrium. From environmental concern, field margin strips buffered boundary vegetation and watercourses against cropped areas loaded with high levels of mineral nitrogen. Margin strips reduced the mineral nitrogen content of the soil in the margin and mineral nitrogen loss during wintermonths. Mineral nitrogen loss was not affected by field margin type but by distance from the field crop. A minimal width of 5 m is necessary to reach an optimal reduction in mineral soil N and N losses.     

Genetic structure of a population sample of apomictic dandelions

In Northern Europe, dandelion populations consist solely of triploid or higher polyploid apomicts. Without a regular sexual cycle or lateral gene transmission, a clonal structure is expected for Taraxacum apomicts, although this was not found by compatibility analysis. In this study, we investigate whether this observation could be suported by performing independent tests based on data from hypervariable microsatellite markers as well as more conservative data based on allozymes and matrilinear cpDNA markers. In addition, population genetic methods were used to test departure from panmictic expectations, which is expected for clonal populations. Results indicated that many data sets, again, did not agree with expectations from clonal evolution because only small groups of genotypes exhibit no marker incompatibility. Population genetic analysis revealed that virtually all genotypes, but not individuals, agreed with random segregation and genotypic equilibria. Exceptions were genotypes with rare allozyme alleles or nearly identical microsatellite genotypes. Consequently, a population sample of apomictic dandelions essentially harbours genotypes that resulted from segregation and/or recombination and only a few genotypes that may have differentiated by somatic mutations.     

Under proper control, oxidation of proteins with known chemical structure provides an accurate and absolute method for the determination of their molar concentration

Oxidation at 120 degrees C of inorganic and organic (including amino acids, di- and tripeptides) model compounds by K(2)Cr(2)O(7) in the presence of H(2)SO(4) (mass fraction: 0.572), Ag(2)SO(4) (catalyst), and HgSO(4) results in the quantitative conversion of their C-atoms into CO(2) within 24 h or less. Under these stressed, well-defined conditions, the S-atoms present in cysteine and cystine residues are oxidized into SO(3) while, interestingly, the oxidation states of all the other (including the N-) atoms normally present in a protein do remain quite unchanged. When the chemical structure of a given protein is available, the total number of electrons the protein is able to transfer to K(2)Cr(2)O(7) and thereof, the total number of moles of Cr(3+) ions which the protein is able to generate upon oxidation can be accurately calculated. In such cases, unknown protein molar concentrations can thus be determined through straightforward spectrophotometric measurements of Cr(3+) concentrations. The values of molar absorption coefficients for several well-characterized proteins have been redetermined on this basis and observed to be in excellent agreement with the most precise values reported in the literature, which fully assesses the validity of the method. When applied to highly purified proteins of known chemical structure (more generally of known atomic composition), this method is absolute and accurate (+/-1%). Furthermore, it is well adapted to series measurements since available commercial kits for chemical oxygen demand (COD) measurements can readily be adapted to work under the experimental conditions recommended here for the protein assay.     

Acceleration of recovery from sciatic nerve damage by the ACTH(4–9) analog Org 2766: Different routes of administration

Functional recovery following a sciatic nerve crush in rats was investigated by measuring the reflex withdrawal of the hindpaw to a hot air stream. The ACTH(4–9) analog Org 2766 accelerated recovery when administered subcutaneously (two-daily injections: 10 μg/animal; minipumps: 20–40 μg/animal per 24 hr; biodegradable microspheres: 40 μg/animal per 24 hr), but oral administration (1.5–20 mg/animal daily, in the drinking water; 1.5–15 mg/animal daily, by gavage) was not effective.     

Survival, Gap Formation, and Recovery Dynamics in Grassland Ecosystems Exposed to Heat Extremes: The Role of Species Richness

A field experiment was performed in which the richness of perennial grasses (S) was varied in model ecosystems exposed to a simulated heat wave (free air temperature increase and drought). The proportion of individuals that survived the heat wave decreased with S, which could be ascribed to higher water consumption in the species-rich systems. Higher transpiration at high diversity was also observed in other studies using functional groups and could have originated from increased leaf area, less intense stomatal closure, or a combination of both. The increased tiller number per plant that we observed, while leaf area per tiller remained constant, suggests that an enhanced leaf area index was most likely responsible. However, competitive interactions also seemed to play a role in the influence of S on survival. Regrowth of the surviving individuals, expressed as leaf area per living plant after a recovery period following the heat wave, increased with S, most likely due to the dominance of productive species, which was facilitated by the additional space yielded by more intense gap formation at higher S (due to higher plant mortality). Species richness affected both the size and density of the gaps. Mean size increased exponentially with S, while density increased at low S but decreased at higher S when connectance of the gaps occurred. Size distribution of the gaps was not affected. Received 18 January 2000; accepted 31 May 2001.     

Quantitative Determination of Polyethylene Glycol Based upon Its Salting Out and Partitioning of a Dye into the Resulting Aqueous Two-Phase System

A method is described that allows quantitative determination of polyethylene glycol (PEG) concentrations by spectrophotometric measurement of fluorescein dye absorbance after its partitioning into an aqueous two-phase system containing mPEG (M_r 5 kDa) in the upper phase and ammonium sulfate in the lower phase. The absorbance decrease of fluorescein in the lower phase is directly proportional to the mPEG concentration, with two proportionality constants equal to 4.42 × 10⁵ and 2.84 × 10⁵ M⁻¹ cm⁻¹ in the range of 0-0.4 and 0.4-1 μM, respectively. This experimental technique can be extended to PEGs of other molecular weights by means of calibration curves that give for each size of PEG the adequate proportionality constants. The results indicate that the quantitative determination is not affected by the presence of many substances such as proteins, reducing agents, and salts, at the usual concentrations.     

Ecology of testate amoebae communities from aquatic habitats in the Zackenberg area (Northeast Greenland)

A total of 67 taxa, belonging to 15 genera, were recorded from aquatic samples collected in Zackenberg (Northeast Greenland). Most taxa belong to the genera Difflugia, Centropyxis, Nebela and Euglypha. Trinema lineare showed the highest relative abundance. Seven taxa were recorded for the first time in the Arctic. A TWINSPAN classification reveals two groups, reflecting a local geographical distinction. In the ponds of the old Zackenberg delta area (group 1), two assemblages were found: the Paraquadrula globulosa-Cyphoderia perlucidus assemblage and the Difflugiella crenulata-Cryptodifflugia compressa assemblage. The lakes, the pools and ponds east of the Zackenberg river (group 2) also harbour two assemblages: the Trinema enchelys-Euglypha sp.1 assemblage and the Centropyxis aerophila-Difflugia globulosa assemblage. Although the lakes are chemically unique, having extremely low values for turbidity, colour, SiO²⁻ ₂, Cl⁻ and hardness, this is not reflected in the characteristic testacean communities. Accepted: 24 April 1999