Beech roots are simultaneously colonized by multiple genets of the ectomycorrhizal fungus Laccaria amethystina clustered in two genetic groups.

In this study, we characterize and compare the genetic structure of aboveground and belowground populations of the ectomycorrhizal fungus Laccaria amethystina in an unmanaged mixed beech forest. Fruiting bodies and mycorrhizas of L. amethystina were mapped and collected in four plots in the Swietokrzyskie Mountains (Poland). A total of 563 fruiting bodies and 394 mycorrhizas were successfully genotyped using the rDNA IGS1 (intergenic spacer) and seven simple sequence repeat markers. We identified two different genetic clusters of L. amethystina in all of the plots, suggesting that a process of sympatric isolation may be occurring at a local scale. The proportion of individuals belonging to each cluster was similar among plots aboveground while it significantly differed belowground. Predominance of a given cluster could be explained by distinct host preferences or by priority effects and competition among genets. Both aboveground and belowground populations consisted of many intermingling small genets. Consequently, host trees were simultaneously colonized by many L. amethystina genets that may show different ecophysiological abilities. Our data showed that several genets may last for at least 1 year belowground and sustain into the next season. Ectomycorrhizal species reproducing by means of spores can form highly diverse and persistent belowground genets that may provide the host tree with higher resilience in a changing environment and enhance ecosystem performance.

Increased inbreeding and strong kinship structure in Taxus baccata estimated from both AFLP and SSR data.

Habitat fragmentation can have severe genetic consequences for trees, such as increased inbreeding and decreased effective population size. In effect, local populations suffer from reduction of genetic variation, and thus loss of adaptive capacity, which consequently increases their risk of extinction. In Europe, Taxus baccata is among a number of tree species experiencing strong habitat fragmentation. However, there is little empirical data on the population genetic consequences of fragmentation for this species. This study aimed to characterize local genetic structure in two natural remnants of English yew in Poland based on both amplified fragment length polymorphism (AFLP) and microsatellite (SSR) markers. We introduced a Bayesian approach that estimates the average inbreeding coefficient using AFLP (dominant) markers. Results showed that, in spite of high dispersal potential (bird-mediated seed dispersal and wind-mediated pollen dispersal), English yew populations show strong kinship structure, with a spatial extent of 50-100 m, depending on the population. The estimated inbreeding levels ranged from 0.016 to 0.063, depending on the population and marker used. Several patterns were evident: (1) AFLP markers showed stronger kinship structure than SSRs; (2) AFLP markers provided higher inbreeding estimates than SSRs; and (3) kinship structure and inbreeding were more pronounced in denser populations regardless of the marker used. Our results suggest that, because both kinship structure and (bi-parental) inbreeding exist in populations of English yew, gene dispersal can be fairly limited in this species. Furthermore, at a local scale, gene dispersal intensity can be more limited in a dense population.

Realized gene flow within mixed stands of Quercus robur L. and Q. petraea (Matt.) L. revealed at the stage of naturally established seedling.

The estimates of contemporary gene flow assessed based on naturally established seedlings provide information much needed for understanding the abilities of forest tree populations to persist under global changes through migration and/or adaptation facilitated by gene exchange among populations. Here, we investigated pollen- and seed-mediated gene flow in two mixed-oak forest stands (consisting of Quercus robur L. and Q. petraea [Matt.] Liebl.). The gene flow parameters were estimated based on microsatellite multilocus genotypes of seedlings and adults and their spatial locations within the sample plots using models that attempt to reconstruct the genealogy of the seedling cohorts. Pollen and seed dispersal were modelled using the standard seedling neighbourhood model and a modification--the 2-component seedling neighbourhood model, with the later allowing separation of the dispersal process into local and long-distance components. The 2-component model fitted the data substantially better than the standard model and provided estimates of mean seed and pollen dispersal distances accounting for long-distance propagule dispersal. The mean distance of effective pollen dispersal was found to be 298 and 463 m, depending on the stand, while the mean distance of effective seed dispersal was only 8.8 and 15.6 m, which is consistent with wind pollination and primarily seed dispersal by gravity in Quercus. Some differences observed between the two stands could be attributed to the differences in the stand structure of the adult populations and the existing understory vegetation. Such a mixture of relatively limited seed dispersal with occasional long distance gene flow seems to be an efficient strategy for colonizing new habitats with subsequent local adaptation, while maintaining genetic diversity within populations.

Extensive pollen flow in two ecologically contrasting populations of Populus trichocarpa.

Pollen-mediated gene flow was measured in two populations of black cottonwood using direct (paternity analysis) and indirect (correlated paternity) methods. The Marchel site was an area with an approximate radius of 250 m in a large continuous stand growing in a mesic habitat in western Oregon. In contrast, the Vinson site was an area with a radius of approximately 10 km and consisted of small, disjunct and isolated stands in the high desert of eastern Oregon. Pollen immigration was extensive in both populations, and was higher in the Marchel site (0.54 +/- 0.02) than in the substantially larger and more isolated Vinson site (0.32 +/- 0.02). Pollen pool differentiation among mothers was approximately five times stronger in the Vinson population (Phi FT = 0.253, N = 27 mothers) than in the Marchel population (Phi FT = 0.052, N = 5 mothers). Pollen dispersal was modelled using a mixed dispersal curve that incorporated pollen immigration. Predicted pollination frequencies generated based on this curve were substantially more accurate than those based on the widely used exponential power dispersal curve. Male neighbourhood sizes (sensu Wright 1946) estimated using paternity analysis and pollen pool differentiation were remarkably similar. They were three to five times smaller in the Vinson population, which reflected the substantial ecological and demographic differences between the two populations. When the same mathematical function was used, applying direct and indirect methods resulted in similar pollen dispersal curves, thus confirming the value of indirect methods as a viable lower-cost alternative to paternity analysis.

Using genetic markers to directly estimate gene flow and reproductive success parameters in plants on the basis of naturally regenerated seedlings.

Estimating seed and pollen gene flow in plants on the basis of samples of naturally regenerated seedlings can provide much needed information about "realized gene flow," but seems to be one of the greatest challenges in plant population biology. Traditional parentage methods, because of their inability to discriminate between male and female parentage of seedlings, unless supported by uniparentally inherited markers, are not capable of precisely describing seed and pollen aspects of gene flow realized in seedlings. Here, we describe a maximum-likelihood method for modeling female and male parentage in a local plant population on the basis of genotypic data from naturally established seedlings and when the location and genotypes of all potential parents within the population are known. The method models female and male reproductive success of individuals as a function of factors likely to influence reproductive success (e.g., distance of seed dispersal, distance between mates, and relative fecundity--i.e., female and male selection gradients). The method is designed to account for levels of seed and pollen gene flow into the local population from unsampled adults; therefore, it is well suited to isolated, but also wide-spread natural populations, where extensive seed and pollen dispersal complicates traditional parentage analyses. Computer simulations were performed to evaluate the utility and robustness of the model and estimation procedure and to assess how the exclusion power of genetic markers (isozymes or microsatellites) affects the accuracy of the parameter estimation. In addition, the method was applied to genotypic data collected in Scots pine (isozymes) and oak (microsatellites) populations to obtain preliminary estimates of long-distance seed and pollen gene flow and the patterns of local seed and pollen dispersal in these species.

Saponins from Hacquetia epipactis.

Four new estersaponins were isolated from hacquetia epipactis. Using GC-MS, FAB-MS and various 2D-NMR techniques they were identified as 3-O-[beta-D-glucopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl-(1--> 3)]- beta-D-glucuronopyranosyl-(1-->)]-21-acetyl-22-(2-methylbutyryl)- barringtogenol C (hacquetiasaponin 1), the corresponding 21-(2-acetoxy-2-methylbutyryl)-22-acetyl-derivative (hacquetiasaponin 2), 3-O-[beta-D-glucopyranosyl-(1-->2)-[alpha-L-arabinopyranosyl- (1-->3)]-beta-D-glucuronopyranosyl-(1-->)]-21-acetyl-22-(2-methylb utyryl)- R1-barrigenol (hacquetiasaponin 3) and its corresponding 21-(2-acetoxy-2-methylbutyryl)-22-acetyl-derivative (hacquetiasaponin 4).

Antimitotic activity of aqueous extracts of Inonotus obliquus.

The cytotoxic effect of two aqueous extracts of Inonotus obliquus on human cervical uteri cancer cells (Hela S3) in vitro was evaluated. It was concluded that Inonotus extracts at a concentration of 10 micrograms/ml to 2000 micrograms/ml inhibited cancer cells growth. In cultures with extracts of the fungus a decrease of the cell proteins and mitotic index was observed. Moreover, the extracts disturbed mitoses by elevating the number of mitotic cells in metaphase. Aqueous extracts of Inonotus effected not only mitoses but also the 8/G phase of the cell cycle.

Time- and dose-dependent antigonadotropic activity of oxidation products of gallic acid and pyrogallol on Leydig cells in vitro.

Auto-oxidation products of plant phenolics in alkaline medium, such as gallic acid and pyrogallol were used to show antigonadotropic activity. The complex mixture of oxidation products was extracted from aqueous medium successively by ethyl ether and ethyl acetate. The fractions obtained were tested on a model of mouse Leydig cells in vitro. All compounds used inhibited luteinizing hormone-stimulated testosterone secretion during 6 and 24 h culture whereas basal secretion was stimulated by pyrogallol oxidation products. Not only low molecular weight substances extracted by organic solvents but also the remaining water soluble, dark brown, high molecular weight products were found to be antigonadotropically active.

Carotenoids in the outer cell-wall layer of Scenedesmus (Chlorophyceae).

Scenedesmus obliquus, strain 633, which synthesizes ketocarotenoids and sporopollenin, also forms pink-red-colored cell walls. Both the cell walls left over after autospore liberation and those from homogenates of disrupted green cells have similar carotenoid pigmentation. Canthaxanthin, astaxanthin, an unidentified ketocarotenoid, and lutein were found as integral cell wall components. They are bound to the outer (trilaminar) layer of the complete cell wall which also contains sporopollenin.

Complex patterns of mating revealed in a Eucalyptus regnans seed orchard using allozyme markers and the neighbourhood model.

The neighbourhood model apportions offspring of individual mother plants to self-fertilization, outcrossing to males within a circumscribed area around the mother plant (the neighbourhood), and outcrossing to males outside the neighbourhood. Formerly the model was applied only to haploid pollen gametes in the offspring of conifers, but is extended so that it can be used with genotypic data from diploid offspring of both angiosperms and gymnosperms. In addition, it is shown that the mating parameters can be estimated without independent estimates of allele frequencies in the pollen pools outside the neighbourhood; thus the model might be applied effectively to natural populations exposed to unknown external pollen sources. Parameters of the neighbourhood mating model were estimated for a 10-year-old seed orchard population of the insect-pollinated tree, Eucalyptus regnans, in southeast Australia, which contained a mixture of two geographical provenances (Victoria and Tasmania). The mating patterns revealed were complex. Crosses between trees of the same provenance occurred three times more often than crosses between trees of different provenances. Levels of self-fertilization and patterns of mating within neighbourhoods were influenced by provenance origin, crop fecundity and orchard position (central vs. edge) of mother trees. Gene dispersal, however, was extensive, with approximately 50% of effective pollen gametes coming from males more than 40 m away from mother trees (average distance between neighbouring trees was 7.4 m). Thus, insect pollinators are efficient promoters of cross-fertilization in this orchard, with the result that the effective number of males mating with each female is large.