Electrophoretic Analysis of Genetic Variation within and between Populations of Quercus cerris,Q. pubescens,Q. petraea and Q. robur (Fagaceae) from Eastern Austria

Allozyme variation at 14 loci was studied electrophoretically in 19 population samples of Quercus cerris, Q. pubescens, Q. petraea, and Q. robur. Genetic variation, measured as mean heterozygosity, was found to be high within populations but low among populations. A significant deficit of heterozygotes was observed in most of the populations of Q. pubescens, Q. petraea, and Q. robur. At the species level, Q. cerris (sect. Cerris) appears to be clearly separated from the rest (sect. Quercus). Q. pubescens, Q. petraea, and Q. robur form a cluster of closely-related species. The degree of genetic differentiation among these species is low, i.e. in the range normally found among con-specific populations. Nevertheless, allozyme data allow the elaboration of a dendrogram which is in reasonable accordance with the taxonomic classification, but also supports the importance of hybridization and introgression. This is also underlined by the fact that the allelic differentiation at the Got-2 locus corresponds with the phenotypic classification (petraea-like, hybrids, and robur-like) in samples from mixed populations.     

Differential response of two Central-European oak species to single and combined stress factors

We studied the effects of repeated defoliation and drought, which are considered to be most effective in triggering oak decline, on the Central-European oak species Quercus robur L. and Quercus petraea [Matt.] Liebl. Three-year-old saplings were subjected to artificial defoliation in two consecutive years and to drought stress, applied singly or in combination. Differences in the morphological, anatomical and physiological responses to these stress factors among treatments and between species were tested in a 3-year experiment. Drought stress lowered the pre-dawn and the afternoon leaf-water potentials in both species, irrespective of additional defoliation. The relative water content of the leaves was unaffected by drought stress; but, in Q. robur, it decreased in trees subjected to defoliation and to the combination of drought stress and defoliation. When repeated defoliation and drought stress were applied within the same growing season, the leaf-to-air difference in the partial pressure of water vapour (w) explained most of the variation in the daily integral of the stomatal conductance to water vapour (g _{s day sum}) in Q. petraea; however, in Q. robur, the hydraulic conductance of the twigs was the most powerful predictor variable. In the same year, the actual hydraulic conductance was correlated with g _{s day sum} in Q. robur, but not in Q. petraea. The studied parameters of gas exchange and biomass production revealed that Q. robur recovered more rapidly from stress than did Q. petraea. We hypothesize that this is due to Q. roburs lower responsiveness to the environmental variable w and its higher responsiveness to internal hydraulic conductance compared to Q. petraea. This revised version was published online in May 2005 with corrections to Table 4.     

Population differentiation of sessile oak at the altitudinal front of migration in the French Pyrenees

To assess the effects of altitude on the level and structure of genetic diversity, a genetic survey was conducted in 12 populations of sessile oak (Quercus petraea) located between 130 and 1660 m in two parallel valleys on the northern side of the Pyrenees Mountains. Genetic diversity was monitored at 16 nuclear microsatellite loci and 5 chloroplast DNA (cpDNA) markers. The cpDNA survey suggested that extant populations in both valleys shared the same source populations from the plain. There was no visible trend of nuclear genetic diversity along altitude, even if indirect estimates of effective population sizes revealed a consistent reduction at higher altitudes. Population differentiation, although low, was mostly present among populations of the same valleys and reached similar levels than differentiation across the range of distribution of sessile oak. Contribution to the overall differentiation in the valleys was mostly due to the genetic divergence of the highest populations and the altitudinal variation of allelic frequencies at a few loci. Bayesian inference of migration between groups of populations showed that gene flow is preferentially unidirectional from lower altitudes in one valley to other groups of populations. Finally, we found evidence of clonal reproduction in high altitude populations. The introgression of Quercus robur and Quercus pubescens was also more frequent at the altitudinal margin suggesting that this mechanism may have contributed to the present migration and adaptation of Q. petraea and may also facilitate its future upslope shift in the context of climate change.     

Bayesian clustering analyses for genetic assignment and study of hybridization in oaks: effects of asymmetric phylogenies and asymmetric sampling schemes

Bayesian clustering methods have been widely used for studying species delimitation and genetic introgression. In order to test the effect of phylogenetic relationships and sampling scheme on the inferred clustering solution and on the performance of Bayesian clustering analysis, I simulated genotypes of the interfertile oak species Quercus robur, Quercus petraea, and Quercus pubescens and I run analyses using two popular software programs, STRUCTURE and BAPS. First, based on purebred simulations, I compared clustering solutions resulting from different sample size configurations. While clustering solution generally reflected the taxonomic relationships when equal samples of each species were included, spurious partition was inferred by STRUCTURE when some species were represented by larger and others by smaller samples. In very unbalanced configurations, STRUCTURE failed to identify the three species, even if three subpopulations were assumed. By contrast, BAPS could properly identify the three species under any sampling scheme. Second, based on simulations of purebreds and hybrids, I tested the performance of individual assignments with variable number of loci. This analysis showed that STRUCTURE can detect introgressed individuals more efficiently than BAPS. However, BAPS could assign purebreds more efficiently with a lower number of loci. Method performance also depended on phylogenetic relationships. In the case of Q. petraea, Q. pubescens, and their hybrids, method performance was lower due to their phylogenetic affinity. Inclusion of three instead of two species into the analysis led to reduction of performance, and to misclassification of hybrids, which often reflected the phylogenetic affinity between Q. petraea and Q. pubescens.     

Ectomycorrhizal fungal communities of pedunculate and sessile oak seedlings from bare-root forest nurseries

In this study, we present the detailed molecular investigation of the ectomycorrhizal (ECM) community of Quercus petraea and Quercus robur seedlings grown in bare-root forest nurseries. In all tested oak samples, mycorrhizal colonization was nearly 100%. Morphological observation and molecular investigations (sequencing of fungal ITS rDNA) revealed a total of 23 mycorrhizal taxa. The most frequent and abundant fungal taxa were Hebeloma sacchariolens, Tuber sp., and Peziza sp.; from the detected fungal taxa, 20 were noted for Q. petraea and 23 for Q. robur. Depending on the nursery, the species richness of identified ECM fungal taxa for both oak species ranged from six to 11 taxa. The mean species richness for all nurseries was 5.36 and 5.82 taxa per Q. petraea and Q. robur sample, respectively. According to the analysis of similarity, ECM fungal communities were similar for Q. petraea and Q. robur (R = 0.019; p = 0.151). On the other hand, detected fungal communities were significantly different between nurseries (R = 0.927; p < 0.0001). Using the Spearman rank correlation, it was determined that the ectomycorrhizal diversity (in terms of richness, the Shannon diversity, evenness, and Simpson dominance indices) is significantly related to the soil parameters of each nursery. We conclude that individual nursery may be considered as separate ecological niches that strongly discriminate diversity of ECM fungi.     

Outlier loci highlight the direction of introgression in oaks

Loci considered to be under selection are generally avoided in attempts to infer past demographic processes as they do not fit neutral model assumptions. However, opportunities to better reconstruct some aspects of past demography might thus be missed. Here we examined genetic differentiation between two sympatric European oak species with contrasting ecological dynamics (Quercus robur and Quercus petraea) with both outlier (i.e. loci possibly affected by divergent selection between species or by hitchhiking effects with genomic regions under selection) and nonoutlier loci. We sampled 855 individuals in six mixed forests in France and genotyped them with a set of 262 SNPs enriched with markers showing high interspecific differentiation, resulting in accurate species delimitation. We identified between 13 and 74 interspecific outlier loci, depending on the coalescent simulation models and parameters used. Greater genetic diversity was predicted in Q. petraea (a late‐successional species) than in Q. robur (an early successional species) as introgression should theoretically occur predominantly from the resident species to the invading species. Remarkably, this prediction was verified with outlier loci but not with nonoutlier loci. We suggest that the lower effective interspecific gene flow at loci showing high interspecific divergence has better preserved the signal of past asymmetric introgression towards Q. petraea caused by the species' contrasting dynamics. Using markers under selection to reconstruct past demographic processes could therefore have broader potential than generally recognized.     

Genetic mapping of EST-derived simple sequence repeats (EST-SSRs) to identify QTL for leaf morphological characters in a Quercus robur full-sib family

The availability of genomic resources such as expressed sequence tag-derived simple sequence repeat (EST-SSR) markers in adaptive genes with high transferability across related species allows the construction of genetic maps and the comparison of genome structure and quantitative trait loci (QTL) positions. In the present study, genetic linkage maps were constructed for both parents of a Quercus robur × Q. robur ssp. slavonica full-sib pedigree. A total of 182 markers (61 AFLPs, 23 nuclear SSRs, 98 EST-SSRs) and 172 markers (49 AFLPs, 21 nSSRs, 101 EST-SSRs, 1 isozyme) were mapped on the female and male linkage maps, respectively. The total map length and average marker spacing were 1,038 and 5.7 cM for the female map and 998.5 and 5.8 cM for the male map. A total of 68 nuclear SSRs and EST-SSRs segregating in both parents allowed to define homologous linkage groups (LG) between both parental maps. QTL for leaf morphological traits were mapped on all 12 LG at a chromosome-wide level and on 6 LG at a genome-wide level. The phenotypic effects explained by each single QTL ranged from 4.0 % for leaf area to 15.8 % for the number of intercalary veins. QTL clusters for leaf characters that discriminate between Q. robur and Quercus petraea were mapped reproducibly on three LG, and some putative candidate genes among potentially many others were identified on LG3 and LG5. Genetic linkage maps based on EST-SSRs can be valuable tools for the identification of genes involved in adaptive trait variation and for comparative mapping.     

Species-specific alleles at a <Emphasis Type="Italic">β-</Emphasis>tubulin gene show significant associations with leaf morphological variation within <Emphasis Type="Italic">Quercus petraea</Emphasis> and <Emphasis Type="Italic">Q. robur</Emphasis> populations

Quercus petraea and Q. robur are largely sympatric oak species in western and central Europe and known for their intensive genetic exchange which has made the discovery of species-diagnostic markers a huge challenge. Various natural white oak populations (Q. petraea/Q. robur including mixed stands) were investigated for their variability and differentiation patterns at a β-tubulin gene (qutub8) in a European-wide survey. This gene was chosen as a possible candidate among loci subjected to selection and maintaining integrity between species. Two frequent alleles depicted as indels within qutub8’s first intron showed remarkably high interspecific genetic differentiation, with Weir and Cockerham’s theta per allele values ranging from 0.17 to 0.30 for one allele and from 0.04 to 0.19 for the other allele in such mixed oak stands where the multi-allelic qutub8 locus showed significant interspecific F _ST . For three mixed stands, qutub8’s F _ST significantly departed from the expected neutral differentiation patterns (F _ST ranging from 0.063 to 0.080 for this multi-allelic marker) and thus could be influenced by selection. Significant associations were found between genotypic variation and leaf dimensions as well as leaf structure patterns, after having accounted for species and stand effects. Qutub8 represents a locus that exhibits significant species differentiation and is linked to morphological discriminant traits. Consequently, qutub8 likely contributes to species divergence within the European white oak complex.     

Flooding effects on starch partitioning during early growth of two oak species

Pedunculate (Quercus robur L.) and sessile (Q. petraea [Matt.] Liebl.) oaks are the most common oak species in Western Europe. They are known to display different ecological requirements, particularly relative to root hypoxia induced by flooding: In a glasshouse study of seedlings, we quantified the effects of flooding on starch mobilization from cotyledons and starch partitioning. Growth and distribution of lateral roots were also measured. The above-ground growth of Q. robur was less affected by flooding than that of Q. petraea which failed to develop a second flush. Root growth was also severely inhibited, particularly in Q. petraea. In Q. robur, lateral root initiation as well as elongation was restricted to the soil surface layer. Flooding markedly reduced total growth and concentrations of in all components except stems. Starch mobilization from cotyledons was delayed by flooding, especially in Q. robur seedlings. Under flooding, the decrease of cotyledons dry mass and starch content in Q. robur was lower than in Q. petraea, whereas Q. robur displayed larger growth than Q. petraea. The features of carbohydrate management may be crucial in the observed differences in flooding tolerance of these species.     

Distinct male reproductive strategies in two closely related oak species

Reproductive strategies of closely related species distributed along successional gradients should differ as a consequence of the trade‐off between competition and colonization abilities. We compared male reproductive strategies of Quercus robur and Q. petraea, two partly interfertile European oak species with different successional status. In the studied even‐aged stand, trees of the late‐successional species (Q. petraea) grew faster and suffered less from intertree competition than trees of the early‐successional species (Q. robur). A large‐scale paternity study and a spatially explicit individual‐based mating model were used to estimate parameters of pollen production and dispersal as well as sexual barriers between species. Male fecundity was found to be dependent both on a tree's circumference and on its environment, particularly so for Q. petraea. Pollen dispersal was greater and more isotropic in Q. robur than in Q. petraea. Premating barriers to hybridization were strong in both species, but more so in Q. petraea than in Q. robur. Hence, predictions based on the competition–colonization trade‐off are well supported, whereas the sexual barriers themselves seem to be shaped by colonization dynamics.     

Hybridisation and colonisation dynamics in European oaks

Summary

Extensive hybridisation between the two sympatric species Quercus petraea and Q.robur is suggested by the near lack of genetic differentiation between the two species and supported by controlled crosses and mating system analysis in mixed stands. Further ecological and genetic evidence suggest that hybridisation does not impede the ecological specialisation of the two species, raising the issue of its evolutionary significance in oaks. Preferential unidirectional hybridization (pollen Q. petraea to ovule Q. robur) has been shown in various mixed stands and facilitates the introduction of sessile oak in existing pedunculate stands. If this unidirectional trend is reinforced in later backcrosses, then hybridisation leads to the dispersal of Q. petraea in existing stands of Q. robur. Hybridisation can therefore be seen as a ‘pollen-mediated’ dispersal mechanism, and has most likely contributed to the rapid migration of Q. petraea in Europe. Given the extant distribution of the species in Europe, migration through pollen swamping should be seen at the edges of the natural distribution of Q. petraea where the demographic imbalance of the two species will reinforce backcrosses.     

Linkage mapping of osmotic stress induced genes of oak

Water stress affecting long-lived trees is an important challenge in forestry. Due to global climate change, forest trees will be threatened by extreme conditions like flooding or drought. It is necessary to understand differences in stress tolerance within certain species and to investigate putative relations on genomic level. In this study, osmotic stress induced genes of Quercus ssp. were positioned on two genetic linkage maps of oak. An intra-specific cross 3P*A4 of Quercus robur consisting of 88 offspring and an inter-specific cross 11P*QS29 of Q. robur and Q. petraea comprising 72 full-sibs were analyzed for the inheritance of 14 loci represented by 34 individual single nucleotide polymorphisms. Seven genes in the intra-cross, as well as other six genes in the inter-cross could be mapped and one gene could not be localised due to the severe distortion of the segregation. The collection of expressed sequences involved ribosomal proteins, members of the oxylase/oxygenase gene family, betaine aldehyde dehydrogenase, Dc3 promoter-binding factor, a putative member of the nodulin family, glutathione-S-transferase and proteins with unknown functions. In the inter-cross, two linked markers exhibited 89% deficiency of heterozygosity. Thirteen genes were positioned on ten different oak chromosomes and can serve as orthologous markers in comparative mapping studies within Fagaceae.     

Changes in the Dynamics of Foliar N Metabolites in Oak Saplings by Drought and Air Warming Depend on Species and Soil Type

Climate change poses direct or indirect influences on physiological mechanisms in plants. In particular, long living plants like trees have to cope with the predicted climate changes (i.e. drought and air warming) during their life span. The present study aimed to quantify the consequences of simulated climate change for foliar N metabolites over a drought-rewetting-drought course. Saplings of three Central European oak species (i.e. Quercus robur, Q. petraea, Q. pubescens) were tested on two different soil types (i.e. acidic and calcareous). Consecutive drought periods increased foliar amino acid-N and soluble protein-N concentrations at the expense of structural N in all three oak species. In addition, transient effects on foliar metabolite dynamics were observed over the drought-rewetting-drought course. The lowest levels of foliar soluble protein-N, amino acid-N and potassium cation with a minor response to drought and air warming were found in the oak species originating from the driest/warmest habitat (Q. pubescens) compared to Q. robur and Q. petraea. Higher foliar osmolyte-N and potassium under drought and air warming were observed in all oak species when grown on calcareous versus acidic soil. These results indicate that species-specific differences in physiological mechanisms to compensate drought and elevated temperature are modified by soil acidity.     

Morphological and physiological reactions of young deciduous trees (Quercus robur L., Q. petraea [Matt.] Liebl., Fagus sylvatica L.) to waterlogging

One-year-old seedlings of Quercus robur L., Q. petraea (Matt.) Liebl. and Fagus sylvatica L. were cultivated in lysimeters and subjected to waterlogging for 17 weeks, interrupted by a five-week drainage period during summer. The growth of Q. robur was less affected by waterlogging than that of Q. petraea and Fagus. Waterlogging resulted in the formation of adventitious roots in Q. robur and Q. petraea, but not in Fagus. In contrast to Fagus, Q. robur and, to a lesser extent, Q. petraea were able to generate roots even below the water table. The hydraulic conductance of the excised root systems, the stomatal conductance and, in Fagus, the leaf water potential and the leaf-mass related hydraulic conductance were decreased by waterlogging. The decrease in the hydraulic conductance was largest in Fagus, and smallest in Q. robur. The roots of Fagus responded to anaerobic conditions with an increase in ethanol concentration. The measurements of nitrate reductase activities in roots and leaves provided no indications of a persistent contribution of NO₃ ⁻ metabolism to the alleviation of waterlogging-induced stress. It is concluded that Q. robur and, to a lesser extent, Q. petraea can tolerate waterlogging periods better than Fagus due to a different pattern of root formation, and to a better adjustment of leaf biomass production to the hydraulic conductivity of the root system. This revised version was published online in June 2006 with corrections to the Cover Date.     

Imprints of Natural Selection Along Environmental Gradients in Phenology-Related Genes of Quercus petraea

We explored single nucleotide polymorphism (SNP) variation in candidate genes for bud burst from Quercus petraea populations sampled along gradients of latitude and altitude in Western Europe. SNP diversity was monitored for 106 candidate genes, in 758 individuals from 32 natural populations. We investigated whether SNP variation reflected the clinal pattern of bud burst observed in common garden experiments. We used different methods to detect imprints of natural selection (F_ST outlier, clinal variation at allelic frequencies, association tests) and compared the results obtained for the two gradients. F_ST outlier SNPs were found in 15 genes, 5 of which were common to both gradients. The type of selection differed between the two gradients (directional or balancing) for 3 of these 5. Clinal variations were observed for six SNPs, and one cline was conserved across both gradients. Association tests between the phenotypic or breeding values of trees and SNP genotypes identified 14 significant associations, involving 12 genes. The results of outlier detection on the basis of population differentiation or clinal variation were not very consistent with the results of association tests. The discrepancies between these approaches may reflect the different hierarchical levels of selection considered (inter- and intrapopulation selection). Finally, we obtained evidence for convergent selection (similar for gradients) and clinal variation for a few genes, suggesting that comparisons between parallel gradients could be used to screen for major candidate genes responding to natural selection in trees.     

Development and Characterization of Genomic and Gene-Based Microsatellite Markers in North American Red Oak Species

Oaks (Quercus: Fagaceae) are ecological and economic keystones of many forested ecosystems but effective genetic management strategies are hindered by high levels of phenotypic plasticity within species and frequent hybridization among them. These same features, however, make oak communities suited for the study of speciation, hybridization, and genetic adaptation. Efforts to develop new and to adapt existing genomic resources to less-studied members of this genus should not only improve oak conservation and management but also aid the study of fundamental evolutionary processes. Here, we present a suite of 27 highly polymorphic simple sequence repeat (SSR) markers tested in four North American red oak (Quercus section Lobatae) species: Q. rubra, Q. ellipsoidalis, Q. coccinea, and Q. velutina. Five markers are genomic SSRs (gSSRs) — four novel and one previously transferred from Q. petraea — and 22 are gene-based SSRs derived from Q. robur and Q. petraea expressed sequence tags (EST-SSRs). Overall, levels of polymorphism detected with these primer pairs were high, with gene diversity (H _e) averaging 0.66 across all loci in natural populations. In addition, we show that EST-SSR markers may have the potential to detect divergent selection at stress-resistance candidate genes among closely related oak species.     

Genetic evidence of reproductive isolation in a remote enclave of Quercus pubescens in the presence of cross-fertile species

Peripheral populations may be crucial for understanding processes underlying adaptive genetic variation. Their evolution and ecology are driven by various genetic and demographic processes, such as selection, gene flow and bottleneck. Peripheral populations often experience a reduction in density resulting in the Allee effect. The presence of interfertile species increases the opportunity for hybridisation, which allows for a rescue from the Allee effect, but at the risk of genetic extinction through introgression. In this article we investigated a peripheral population of Quercus pubescens, a European tree species. The study population is located in Poland, several hundred kilometres northwards from the main species range. Due to geographic separation, the study population exists under strong pressure of introgression from potentially inter-fertile Q. petraea and Q. robur, which are the only common oaks in Poland. The intermediate morphology between typical Q. pubescens and a common oak species found in the study population supports the introgression hypothesis, which could be in line with the earlier studies of this species complex conducted in the main range of Q. pubescens. Alternatively, the intermediate morphology could reflect the founder effect or selection at an ecological extreme. We attempted to verify these hypotheses using microsatellites and a reference of common oak species. The results showed that the study population is genetically distinct from both Q. petraea and Q. robur. Additionally, the population is characterised by a low effective population size and limited gene dispersal. This suggests that the study population reveals strong reproductive isolation from common species, implying alternative sources of atypical morphology.     

Development of ten microsatellite markers for <Emphasis Type="Italic">Quercus mongolica</Emphasis> var. <Emphasis Type="Italic">crispula</Emphasis> by database mining

Simple sequence repeats (SSRs) in the NCBI dbEST database were surveyed to identify potential SSR markers for Quercus mongolica. In total, 2,691 gene sequences, mainly from expressed sequence tags (ESTs) for Q. robur and Q. petraea had been registered. Twenty-two PCR primers were designed for SSRs in these sequences and screened for polymorphisms in 16 Q. mongolica trees. Ten loci were easily genotyped and showed polymorphism, with numbers of alleles and expected heterozygosity ranging from 3 to 15 and 0.28 to 0.94, respectively. These EST-SSR markers should be useful for studying the genetic diversity of Quercus species.     

Utility of multilocus genotypes for taxon assignment in stands of closely related European white oaks from Switzerland

Background and Aims

European white oaks (Quercus petraea, Q. pubescens, Q. robur) have long puzzled plant biologists owing to disputed species differentiation. Extensive hybridization or shared ancestry have been proposed as alternative hypotheses to explain why genetic differentiation between these oak species is low. Species delimitation is usually weak and often shows gradual transitions in leaf morphology. Hence, individual identification may be difficult, but remains a critical step for both scientific work and practical management.

Methods

Multilocus genotype data (five nuclear microsatellites) were used from ten Swiss oak stands for taxon identification without a priori grouping of individuals or populations, using model-based Bayesian assignment tests.

Key Results

Three groups best structured the data, indicating that the taxonomical signal was stronger than the spatial signal. Most individuals showed high posterior probabilities for either of three genetic groups that were best circumscribed as taxonomical units. The assignment of a subset of trees, whose taxonomic status had been previously characterized in detail, supported this classification scheme.

Conclusions

Molecular-genetic assignment tests are useful in the identification of species status in critical taxon complexes such as the European white oaks. Such an approach is of practical importance for forest management, e.g. for stand certification or in seed trade to trace the origin of forest products.Key words: Assignment test, Bayesian inference, multilocus genotype, nuclear microsatellites, Quercus sp., species complex