Assessment of genetic structure within and among Bulgarian populations of the common ash (Fraxinus excelsior L.)

We analysed genetic variation within and between populations of the common ash from Bulgaria in order to extract biological information useful in the context of conservation management of eastern European genetic resources of noble hardwood species. A total of 321 trees from three regions of Bulgaria were typed at six highly polymorphic microsatellite loci. Analysis of within-population inbreeding suggests an upper boundary value of 2.7% for the selfing rate. Significant spatial genetic structure consistent with models of isolation by distance was detected within four out of 10 populations as well as among populations. Estimates of neighbourhood size in the range 38-126 individual trees were obtained based on spatial genetic structure analyses at either the intrapopulation or interpopulation level. Differentiation among populations explained only about 8.7% of total genetic diversity. These results are discussed in comparison with data from social broad-leaved species such as oak and beech.     

Molecular biodiversity and population structure in common ash (Fraxinus excelsior L.) in Britain: implications for conservation

Current forestry policy promotes the use of local seed for new plantings, on the assumption that local material may be better adapted to local conditions. However, landscape‐scale genetic studies which are necessary to underpin conservation and breeding strategies are often lacking. We investigated molecular diversity in common ash (Fraxinus excelsior L.) sampled from 42 British and six French sites with microsatellites. Chloroplast haplotype H04 was the most common and widespread in Britain, although rare and localized individuals with H02 and H09 were also detected. In addition, three new chloroplast haplotypes were identified, and these were rare and highly localized. In terms of nuclear microsatellite markers, allelic richness differed between sites and decreased in an east to west direction. Differentiation between sites was often very low (mean F_ST 0.025), indicating few differences between the majority of sites. There was a clear excess of homozygotes (mean H_O 0.669, mean H_E 0.818) and a relatively high F_IS (mean 0.182), suggests a consistent level of inbreeding or a widespread Wahlund effect in many F. excelsior sites. Gene pool ancestry analysis suggested that the majority of British F. excelsior belongs to a single meta‐population which covers mainland western and central Europe. Three northern and western sites diverged markedly from the dominant population, and may represent remnants of two late potential Ice Age refugia in northern Britain. The data provide new information which will aid development of appropriate conservation policies for ash and other wind pollinated tree species.     

Chloroplast DNA variation and postglacial recolonization of common ash (Fraxinus excelsior L.) in Europe

We used chloroplast polymerase chain reaction-restriction-fragment length polymorphism (PCR-RFLP) and chloroplast microsatellites to assess the structure of genetic variation and postglacial history across the entire natural range of the common ash (Fraxinus excelsior L.), a broad-leaved wind-pollinated and wind-dispersed European forest tree. A low level of polymorphism was observed, with only 12 haplotypes at four polymorphic microsatellites in 201 populations, and two PCR-RFLP haplotypes in a subset of 62 populations. The clear geographical pattern displayed by the five most common haplotypes was in agreement with glacial refugia for ash being located in Iberia, Italy, the eastern Alps and the Balkan Peninsula, as had been suggested from fossil pollen data. A low chloroplast DNA mutation rate, a low effective population size in glacial refugia related to ash's life history traits, as well as features of postglacial expansion were put forward to explain the low level of polymorphism. Differentiation among populations was high (GST= 0.89), reflecting poor mixing among recolonizing lineages. Therefore, the responsible factor for the highly homogeneous genetic pattern previously identified at nuclear microsatellites throughout western and central Europe (Heuertz et al. 2004) must have been efficient postglacial pollen flow. Further comparison of variation patterns at both marker systems revealed that nuclear microsatellites identified complex differentiation patterns in south-eastern Europe which remained undetected with chloroplast microsatellites. The results suggest that data from different markers should be combined in order to capture the most important genetic patterns in a species.     

Underwater germination potential of common ash seed (Fraxinus excelsior L.) originating from flooded and non-flooded sites

Oxygen availability is one of the main factors affecting seed germination capacity. On floodplains, oxygen supply is seasonally restricted because of flooding events. Seed ecotypes are able to cope with total submergence to different extents, depending on the species and its position in a given environmental gradient. This investigation tested three provenances of common ash (Fraxinus excelsior L.), one from a lowland site and two from upland sites, for germination capacity and germination velocity under water. The final germination capacity of all provenances was unaffected, but the germination velocity was decreased for all provenances. The adaptational significance of these results is discussed.     

Micropropagation of hornbeam (Carpinus betulus L.) and ash (Fraxinus excelsior L.)

Shoot multiplication of hornbeam was stimulated onWPM, QL andDKW medium supplemented with a low concentration of BAP or BPA (0.1-0.2 mg I -1) andIBA (0.1 mg I -1). Low concentration of thidiazuron promoted axillary bud formation, higher concentration inhibited shoot elongation. Microshoots were rooted onWPM supplemented with a low auxin concentration (IBA or NAA 0.2-0.5 mg I -1). High rooting percentages were obtained. Shoot proliferation of ash was stimulated on MS andDKW medium supplemented withBAP orBPA (2.0-5.0 mg I -1) andIBA (0.1 mg I -1). Root formation was promoted onWPM containing a low auxin concentration. Rooted plantlets were transplanted into soil and after hardening off the micropropagated trees were planted in the field. The planted trees grew normally without showing signs of abnormality.     

Genetic variability of Chalara fraxinea, dieback cause of European ash (Fraxinus excelsior L.)

A total of 159 colonies of Chalara fraxinea were isolated between 2005 and 2006 from dying trees of European ash (Fraxinus excelsior L.) aged between 3 and 10 years. They derived from five regions of Poland differing by geographic location and climatic conditions. On the basis of 90 RAMS markers, pathogen intra- and inter-population variability, as well as its dependency on geographic distance and climatic conditions in the regions of strain origin, was analysed. The applied measures of intrapopulation genetic variability (genetic distance, Nei’s unbiased diversity, Shannon’s Information Index and percentage of polymorphic loci) allowed for differentiation of two strain groups: the first deriving from lowlands and the second from uplands and mountainous areas. Strains in lowlands were characterised by smaller number of markers, smaller number of polymorphic loci and smaller intrapopulation genetic variability. Positive and statistically significant correlation was shown between variability of isolates and elevation of regions above sea level. Pair-wise genetic distances between groups of isolates (Nei’s unbiased genetic distance) from particular regions were not significantly correlated with the corresponding geographic distances. On the basis of AMOVA, it was shown that 85% of variability was within-region differences and 2% between-region differences, whereas differences between lowlands and uplands were 13%. Principal Components Analysis (PCA) for the investigated regions confirmed the results from Nei’s genetic distance matrix.     

Molecular characterization of Serratia marcescens strains by RFLP and sequencing of PCR-amplified 16S rDNA and 16S-23S rDNA intergenic spacer

AIMS: To establish the specific DNA patterns in 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) regions from different kinds of Serratia marcescens strains using polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP) and sequences analysis. METHODS AND RESULTS: Two pairs of primers based on the 16S rDNA and 16S-23S rDNA IGS were applied to amplify the rrn operons of two kinds of S. marcescens strains. About 1500 bp for 16S rDNA and four fragments of different sizes for 16S-23S rDNA IGS were obtained. PCR-amplified fragments were analysed by RFLP and sequence analysis. Two distinct restriction patterns revealing three to five bands between two kinds of strains were detected with each specific enzyme. According to the sequence analysis, two kinds of strains showed approximately 97% sequence homology of 16S rDNA. However, there was much difference in the sequences of IGS between the two kinds of strains. Intercistronic tRNA of strains H3010 and A3 demonstrated an order of tRNA of 5'-16S-tRNA(Ala)-tRNA(Ile)-23S-3', but strain B17 harboured the tRNA of 5'-16S-tRNA(Glu)-tRNA(Ile)-23S-3'. CONCLUSIONS: The method was specific, sensitive and accurate, providing a new technique for differentiating different strains from the same species. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provided the first molecular characterization of 16S rDNA and 16S-23S rDNA IGS from S. marcescens strains.     

Utility of the polymerase chain reaction-restriction fragment length polymorphisms of the intergenic spacer region of the rDNA for characterizing Gibberella fujikuroi isolates

In the present report, a total of thirty-one isolates of Gibberella fujikuroi (Sawada) Wollenw. species complex of Fusarium (section Liseola) morphologically classified as F. moniliforme according to the taxonomy of Nelson, Toussoun and Marasas (1983) were analyzed for their ability to produce fumonisin B₁ and fumonisin B₂ by an optimized liquid chromatographic method. They were isolated from three hosts (Zea mays, Musa sapientum and Pinus pinea). The results indicate that M. sapientum is a preferential host for G. fujikuroi isolates with low or null capacity for producing fumonisins, while isolates from Z. mays and P. pinea are generally high fumonisin producers.

The molecular characterization of isolates was carried out in parallel using an optimized, simple and low-cost method for isolating DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the rDNA intergenic spacer (IGS) region. The haplotypes obtained with Hha I enzyme and combinations of Hha I, EcoR I, Alu I, Pst I and Xho I enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin B₁ and B₂ producing capacity. IGS region restriction patterns showed no relationship to isolate geographical origin. This is the first report on this method's capacity to detect polymorphism permitting discrimination between G. fujikuroi isolates from different hosts and with different toxigenic profiles.     

Nuclear microsatellites reveal contrasting patterns of genetic structure between western and southeastern European populations of the common ash (Fraxinus excelsior L.)

To determine extant patterns of population genetic structure in common ash and gain insight into postglacial recolonization processes, we applied multilocus-based Bayesian approaches to data from 36 European populations genotyped at five nuclear microsatellite loci. We identified two contrasting patterns in terms of population genetic structure: (1) a large area from the British Isles to Lithuania throughout central Europe constituted effectively a single deme, whereas (2) strong genetic differentiation occurred over short distances in Sweden and southeastern Europe. Concomitant geographical variation was observed in estimates of allelic richness and genetic diversity, which were lowest in populations from southeastern Europe, that is, in regions close to putative ice age refuges, but high in western and central Europe, that is, in more recently recolonized areas. We suggest that in southeastern Europe, restricted postglacial gene flow caused by a rapid expansion of refuge populations in a mountainous topography is responsible for the observed strong genetic structure. In contrast, admixture of previously differentiated gene pools and high gene flow at the onset of postglacial recolonization of western and central Europe would have homogenized the genetic structure and raised the levels of genetic diversity above values in the refuges.     

Nitrate reduction and nitrate content in ash trees (Fraxinus excelsior L.): distribution between compartments,site comparison and seasonal variation

Summary Nitrate reductase activity (NRA), nitrate content and biomass components of leaflets, leaf stalks, old stem, current-year stem and roots of ash trees (Fraxinus excelsior L.) growing in their natural habitats were investigated. In addition, NRA, total nitrogen and nitrate concentration were analyzed in the leaves and roots of ash trees from four different field sites. The highest NRA per gram biomass and also per total compartment biomass was found in the leaflets, even though root biomass was much higher than total leaflet biomass. The highest nitrate concentrations were found in the leaf stalks. Correlations between nitrate availability in the soil and NRA in leaves were not significant due to high variability of the actual soil nitrate concentrations. The seasonal variation in foliar NRA, nitrate concentration and total nitrogen concentration is much smaller in F. excelsior than reported for herbaceous species and is mainly caused by changes in the actual soil nitrate availability and by senescence of the leaves.     

Cumulative temperatures for prediction of the beginning of ash (Fraxinus excelsior L.) pollen season

The aim of this study is the analysis of the meteorological conditions leading to the release of ash pollen in order to predict the beginning of the pollen season in Zurich, Switzerland. For 12 years over a period of 14 consecutive years, it appears that ash pollen release begins when the cumulated mean daily temperatures starting on January 1 reach 220.7°C, admitting a variation of ±30°C that more or less corresponds to 3 consecutive days at 10°C.     

Changes in ecosystem carbon stocks in a grassland ash (Fraxinus excelsior) afforestation chronosequence in Ireland

Aims Government policy in Ireland is to increase the national forest cover from the current 10% to 18% of the total land area by 2020. This represents a major land use change that is expected to impact on the national carbon (C) stocks. While the C stocks of ecosystem biomass and soils of Irish grasslands and coniferous forests have been quantified, little work has been done to assess the impact of broadleaf afforestation on C stocks.Methods In this study, we sampled a chronosequence of ash (Fraxinus excelsior) forests aged 12, 20, 27, 40 and 47 years on brown earth soils. A grassland site, representative of the pre-afforestation land use, was sampled as a control.Important findings Our results show that there was a significant decline (P < 0.05) in the carbon density of the soil (0–30cm) following afforestation from the grassland (90.2 Mg C ha^-1) to the 27-year-old forest (66.7 Mg C ha^-1). Subsequently, the forest soils switched from being a C source to a C sink and began to sequester C to 71.3 Mg C ha^-1 at the 47-year-old forest. We found the amount of C stored in the above- and belowground biomass increased with age of the forest stands and offset the amount of C lost from the soil. The amount of C stored in the above- and belowground biomass increased on average by 1.83 Mg C ha^-1 year^-1. The increased storage of C in the biomass led to an increase in the total ecosystem C, from 90.2 Mg C ha^-1 at the grassland site to 162.6 Mg C ha^-1 at the 47-year-old forest. On a national scale, projected rates of ash afforestation to the year 2020 may cause a loss of 290 752 Mg C from the soil compared to 2 525 936 Mg C sequestered into the tree biomass. The effects of harvesting and reforestation may further modify the development of ecosystem C stocks over an entire ash rotation.