Genetic variation and differentiation within a natural community of five oak species (Quercus spp.)

Chloroplast DNA and two categories of nuclear markers - isozymes and microsatellites - were used to examine a very rich natural community of oaks (Quercus spp.) situated in west-central Romania. The community consists of five oak species: Q. robur, Q. petraea, Q. pubescens, and Q. frainetto - that are closely related -, and Q. cerris. A total of five chloroplast haplotypes was identified. Q. cerris was fixed for a single haplotype. The other four species shared the two most common haplotypes. One haplotype was confined to Q. robur and a very rare one was restricted to Q. petraea. Both types of nuclear markers revealed a larger genetic variation for Q. pubescens and Q. petraea than for Q. frainetto and Q. robur, although the differences between species are in most cases not significant. At the nuclear level, Q. cerris could be clearly separated from the other four oak species confirming the taxonomic classification. Regardless of the estimate used, the levels of polymorphism revealed by microsatellites were much higher than those based on isozymes. For the four closely related species the overall genetic differentiation was significant at both categories of nuclear markers. Several loci, such as Acp-C for isozymes, and ssrQpZAG36 and ssrQrZAG96 for microsatellites were very useful to discriminate among species. However, the level of differentiation varied markedly between pairs of species. The genetic affinities among the species may reflect different phylogenetic distances and/or different rates of recurrent gene flow at this site.     

The Description of Clones of Quercus robur L. and Q. petraea (Matt.) Liebl. with Microsatellites and AFLP in an Ancient Woodland

Abstract: In densely populated areas autochthonous Quercus robur L. (pedunculate oak) and Q. petraea (Matt.) Liebl. (sessile oak) (Fagaceae) populations have been maintained as ancient devastated woodlands. The continuous cutting, grazing and resprouting of such woodlands has enabled the development of clonal structures. For conservation purposes, an analysis of the actual number, size and spatial distribution of clones is necessary, especially when there is an interest in genetic variation of the population. This study describes for the first time - based on microsatellite and AFLP™ analysis - clones in an autochthonous mixed Q. robur and Q. petraea population that has been coppiced and grazed for several centuries. Based on six microsatellite loci and 69 polymorphic AFLP markers, only 14 unique genotypes were detected in a plot that consisted of 80 trees. Clones were observed for both Q. robur and Q. petraea. The largest clone diameters were observed for Q. robur, with distances up to 5.8 m. The observed clone sizes may indicate the age of the trees.     

Identification and characterization of (GA/CT) n - microsatellite loci from Quercus petraea

In this study a size selected genomic library from Quercus petraea was screened for (GA/CT)_n-microsatellite sequences. The resulting loci were analysed by PCR for their usefulness as molecular markers in Q. petraea and Q. robur. 17 out of 52 tested primer pairs resulted in the amplification of a polymorphic single-locus pattern. The number of alleles found per locus varied from 6 to 16. Combining the genetic variation observed for the characterized loci provides a unique genotype for all the individuals tested. Using intraspecific controlled crosses of Q. robur trees Mendelian inheritance could be shown for five loci.     

Natural hybridisation between Quercus petraea (Matt.) Liebl. and Quercus pubescens Willd. within an Italian stand as revealed by microsatellite fingerprinting

Interspecific gene flow is frequently reported in the genus Quercus . However, interfertile oak species often seem to remain distinct, even within areas of sympatry. This study employed molecular markers to verify, at a fine scale, the presence of interspecific gene flow in a natural population of Quercus petraea and Quercus pubescens . Within a delimited area of 6 ha, all adult trees belonging to the studied oak complex and seeds from a subsample of such trees were collected and analysed using molecular microsatellite markers. A low interspecific genetic differentiation and a high level of interspecific genetic admixture suggested past hybridisation. Paternity inference of seeds allowed the estimation of pollination frequencies from the three groups of pollen donors ( Q. petraea, Q. pubescens , intermediate). We also assayed pollen viability and germinability of each species group. We observed natural hybridisation between Q. petraea and Q. pubescens, with a predominant component in the direction Q. petraea → Q. pubescens : Q. pubescens displayed a higher level of heterospecific pollination by Q. petraea (25.8%) and intermediate morphotypes (14.7%), compared to Q. petraea acting as pollen receptor (with less than 5% heterospecific pollinations). Intermediate 'mother trees' were pollinated in similar proportions by Q. petraea (23.1%), Q. pubescens (37.8%) and intermediate morphotypes (39.1%). The asymmetrical introgression observed for the studied generation may be caused, among other factors, by the relative abundance of trees from each species group in the studied area.     

QTL analysis of leaf morphological characters in a Quercus robur full-sib family (Q. robur x Q. robur ssp. slavonica)

The distinction between white oak species (section Quercus sensu stricto) is largely based on leaf morphological characters. There is, however, considerable within-species variation and no single species-diagnostic character, possibly due to phenotypic plasticity and/or underlying genetic variation. The aim of the present study was to identify quantitative trait loci (QTL) underlying the high within-species variation for leaf morphological characters in an F(1) full-sib family derived from a cross between Q. robur and Q. robur ssp. slavonica. In accordance with an earlier QTL mapping study in an intraspecific Q. robur full-sib family, polygenic inheritance was detected for leaf morphological characters that are used to discriminate between the species Quercus robur and Q. petraea. QTLs were distributed over ten linkage groups, showed a moderate effect in terms of phenotypic variance explained (PVE) in the mapping pedigree (3.6-9.6%), but accounted for a considerable amount of the parental differences. Co-localisation of QTLs on the same linkage group in different genetic backgrounds was found for the number and percentage of intercalary veins (NV, PV) on linkage group 3 and for NV on linkage group 5, revealing a high congruence in the relative QTL positions. The generally low correspondence of the other QTLs in the different mapping pedigrees may be an effect of the genetic background and of the environment. In conclusion, leaf morphological characters were found to be under polygenic control, and a comparison to earlier published results led to the identification of two QTLs that were stable across different genetic backgrounds.     

QTL analysis of leaf morphological characters in a Quercus robur full-sib family (Q. robur × Q. robur ssp. slavonica)

The distinction between white oak species (section Quercus sensu stricto ) is largely based on leaf morphological characters. There is, however, considerable within-species variation and no single species-diagnostic character, possibly due to phenotypic plasticity and/or underlying genetic variation. The aim of the present study was to identify quantitative trait loci (QTL) underlying the high within-species variation for leaf morphological characters in an F₁ full-sib family derived from a cross between Q. robur and Q. robur ssp. slavonica . In accordance with an earlier QTL mapping study in an intraspecific Q. robur full-sib family, polygenic inheritance was detected for leaf morphological characters that are used to discriminate between the species Quercus robur and Q. petraea . QTLs were distributed over ten linkage groups, showed a moderate effect in terms of phenotypic variance explained (PVE) in the mapping pedigree (3.6–9.6%), but accounted for a considerable amount of the parental differences. Co-localisation of QTLs on the same linkage group in different genetic backgrounds was found for the number and percentage of intercalary veins (NV, PV) on linkage group 3 and for NV on linkage group 5, revealing a high congruence in the relative QTL positions. The generally low correspondence of the other QTLs in the different mapping pedigrees may be an effect of the genetic background and of the environment. In conclusion, leaf morphological characters were found to be under polygenic control, and a comparison to earlier published results led to the identification of two QTLs that were stable across different genetic backgrounds.     

Species distinction in Irish populations of Quercus petraea and Q. robur: morphological versus molecular analyses

BACKGROUND AND AIMS: Populations of oak (Quercus petraea and Q. robur) were investigated using morphological and molecular (AFLP) analyses to assess species distinction. The study aimed to describe species distinction in Irish oak populations and to situate this in a European context. METHODS: Populations were sampled from across the range of the island of Ireland. Leaf morphological characters were analysed through clustering and ordination methods. Putative neutral molecular markers (AFLPs) were used to analyse the molecular variation. Cluster and ordination analyses were also performed on the AFLP markers in addition to calculations of genetic diversity and F-statisitcs. KEY RESULTS: A notable divergence was uncovered between the morphological and molecular analyses. The morphological analysis clearly differentiated individuals into their respective species, whereas the molecular analysis did not. Twenty species-specific AFLP markers were observed from 123 plants in 24 populations but none of these was species-diagnostic. Principal Coordinate Analysis of the AFLP data revealed a clustering, across the first two axes, of individuals according to population rather than according to species. High F(ST) values calculated from AFLP markers also indicated population differentiation (F(ST) = 0.271). Species differentiation accounted for only 13 % of the variation in diversity compared with population differentiation, which accounted for 27 %. CONCLUSIONS: The results show that neutral molecular variation is partitioned more strongly between populations than between species. Although this could indicate that the populations of Q. petraea and Q. robur studied may not be distinct species at a molecular level, it is proposed that the difficulty in distinguishing the species in Irish oak populations using AFLP markers is due to population differentiation masking species differences. This could result from non-random mating in small, fragmented woodland populations. Hybridization and introgression between the species could also have a significant role.     

Gene flow and hybridisation in a mixed oak forest (Quercus pyrenaica Willd. and Quercus petraea (Matts.) Liebl.) in central Spain

Oaks are long-standing models for the study of gene flow and hybridisation. Temperate (Quercus petraea) and sub-Mediterranean (Quercus pyrenaica) oaks coexist in central Spain, showing remarkable differences in population size and structure. Q. petraea has a scattered distribution in central Spain, where it is at one of the southernmost limits of its range, and forms low-density stands; in contrast, Q. pyrenaica is widespread in the region. We selected a mixed population of the two species ( approximately 13 ha, 176 adults and 96 saplings) to compare the patterns of gene flow within each species and the extent of introgression between them. Using five nuclear microsatellite markers, we performed a parentage analysis and found considerable immigration from outside the stand ( approximately 38% for Q. petraea and approximately 34% for Q. pyrenaica), and estimated average seed-dispersal distances of 42 and 14 m for Q. petraea and Q. pyrenaica, respectively. Introgression between species was also estimated using our microsatellite battery. First, we developed a multivariate discriminant approach and, second, we compared our results with a widely used clustering method (STRUCTURE). Both analyses were consistent with a low level of introgression between Q. petraea and Q. pyrenaica. Indeed, only 15 adult trees, approximately 8.5%, were identified as putative hybrids when both methods of analysis were combined. Hybrids may be most common in contact zones due merely to physical proximity.     

Pollen dispersal inferred from paternity analysis in a mixed oak stand of Quercus robur L. and Q. petraea (Matt.) Liebl.

Paternity analysis was used to determine the spatial distribution of male parents of 984 offspring collected from 13 identified mother trees in a natural stand of 5.76 ha and comprising 296 adult trees of Quercus petraea and Q . robur . For seven of the 13 maternal progeny arrays sampled, we found an excess of nearby matings and a preferential direction of pollination. For the remaining progeny arrays, no departure from random distribution of male parents was detected. A common trend among all families was a high percentage (averaging 65% for Q. robur and 69% for Q. petraea ) of offspring that were pollinated by male parents from outside the study site. By pooling the data over all families, the average pollen dispersal curve within the stand was inferred and fitted to a negative exponential distribution. This model extrapolated for distances over the spatial scale of the study stand was insufficient to explain the high level of gene flow detected by the paternity analysis, suggesting a substantial level of long-distance pollination events. The genetic composition of the pollen pools received by each maternal tree was compared and showed significant differentiation that could be attributed to differences in male reproductive success. By contrast, no significant differentiation between the pollen clouds from outside and inside the study stand was detected, suggesting genetic homogeneity between the surrounding forest and the study stand.     

Frequent cytoplasmic exchanges between oak species that are not closely related: Quercus suber and Q. ilex in Morocco

Chloroplast (cp) and mitochondrial (mt) DNA variation were studied in 97 populations of cork oak (Quercus suber) in Morocco; in 31 of these populations, holm oak (Quercus ilex), a clearly distinct species, also occurred and was compared with Q. suber. Three cpDNA and one mtDNA primer pairs were used in the survey, each in combination with one restriction enzyme. Six haplotypes belonging to two very divergent lineages were detected; one lineage predominates in each species, and is probably ancestral, as inferred from comparisons with other oak species. In the mixed-species populations, cytoplasmic genomes were frequently shared across species, as indicated by an introgression ratio of 0.63. This index is a new measure of the propensity of species to share locally genetic markers, varying from zero (complete differentiation) to one (no differentiation). By contrast, more closely related deciduous oak species (Q. robur, Q. petraea and Q. pubescens) have introgression ratios varying from 0.82 to 0.97. The introgression events appear to have been more frequent in the direction Q. ilex (female) x Q. suber (male), a finding which seems attributable to the flowering phenology of these two species. This asymmetry may have favoured immigration of Q. suber beyond its main range, in regions already colonized by Q. ilex. There, rare hybridization and further introgression through long distance pollen flow have established populations that are morphologically indistinguishable from Q. suber but that have cytoplasmic genomes originating from the local Q. ilex populations.     

Results on quantitative trait loci for flushing date in oaks can be transferred to different segregating progenies

Flushing date (bud burst) is one of the most important traits for the adaptation to different environments and climates in the temperate zone. Because of their wide geographic distribution, Quercus robur L. and Q. petraea (Matt.) Liebl. are suitable as model plants to study the genetic basis of bud burst. QTLs (Quantitative Trait Loci) with comparatively large effects have been mapped in a former study in a Q. robur x Q. robur full-sib family (French cross). In the present study, we performed a Bulked Segregant Analysis (BSA) in the F (1) progeny comprising 144 seedlings derived from a cross between a single Q. robur tree as common seed parent and five different pollen donors both from Q. robur and Q. petraea (Q. robur x Q. spp., Diekholzen crosses). In addition, markers linked to two bud burst QTLs with comparably strong effect in the above-mentioned full-sib family (French cross) were tested for their association with bud burst in the Q. robur x Q. spp. (Diekholzen) progeny. Using three microsatellite markers as anchor points, we could map QTLs on linkage group 7 and on linkage group 2, together explaining 16.2 % of the total phenotypic variance (PVE) in 1999 and 38.1 % in 2003. Out of 10 markers that segregated in both mapping progenies, four markers including the two microsatellite markers, showed a significant effect on bud burst in both materials. At microsatellite loci ssrQpZAG1/5 (linkage group 7) and ssrQpZAG119 (linkage group 2) alleles associated with early (allele 166 bp in ssrQpZAG1/5) and late bud burst (allele 57 bp in ssrQpZAG119) in the Q. robur x Q. robur full-sib family (French cross) showed a highly significant association with the same polarity of the effect in the Q. robur x Q. spp. (Diekholzen) progeny. The usefulness of these markers for marker-assisted selection in full-sib and half-sib families is discussed.     

Evidence for shared ancestral polymorphism rather than recurrent gene flow at microsatellite loci differentiating two hybridizing oaks (Quercus spp.)

Quercus petraea and Quercus robur are two closely related oak species, considered to hybridize. Genetic markers, however, indicate that despite sharing most alleles, the two species remain separate genetic units. Analysis of 20 microsatellite loci in multiple populations from both species suggested a genome-wide differentiation. Thus, the allele sharing between both species could be explained either by low rates of gene flow or shared ancestral variation. We performed further analyses of population differentiation in a biogeographical setting and an admixture analysis in mixed oak stands to distinguish between both hypotheses. Based on our results we propose that the low genetic differentiation among these species results from shared ancestry rather than high rates of gene flow.     

Strength, diversity and plasticity of postmating reproductive barriers between two hybridizing oak species (Quercus robur L. and Quercus petraea (Matt) Liebl.)

Very little is known about the nature and strength of reproductive isolation (RI) in Quercus species, despite extensive research on the estimation and evolutionary significance of hybridization rates. We characterized postmating pre- and postzygotic RI between two hybridizing oak species, Quercus robur and Quercus petraea, using a large set of controlled crosses between different genotypes. Various traits potentially associated with reproductive barriers were quantified at several life history stages, from pollen-pistil interactions to seed set and progeny fitness-related traits. Results indicate strong intrinsic postmating prezygotic barriers, with significant barriers also at the postzygotic level, but relatively weaker extrinsic barriers on early hybrid fitness measures assessed in controlled conditions. Using general linear modelling of common garden data with clonal replicates, we showed that most traits exhibited important genotypic differences, as well as different levels of sensitivity to micro-environmental heterogeneity. These new findings suggest a large potential genetic diversity and plasticity of reproductive barriers and are confronted with hybridization evidence in these oak species.     

Genome scanning for interspecific differentiation between two closely related oak species [Quercus robur L. and Q. petraea (Matt.) Liebl.

Interspecific differentiation values (G(ST)) between two closely related oak species (Quercus petraea and Q. robur) were compiled across different studies with the aim to explore the distribution of differentiation at the genome level. The study was based on a total set of 389 markers (isozymes, AFLPs, SCARs, microsatellites, and SNPs) for which allelic frequencies were estimated in pairs of populations sampled throughout the sympatric distribution of the two species. The overall distribution of G(ST) values followed an L-shaped curve with most markers exhibiting low species differentiation (G(ST) < 0.01) and only a few loci reaching >10% levels. Twelve percent of the loci exhibited significant G(ST) deviations to neutral expectations, suggesting that selection contributed to species divergence. Coding regions expressed higher differentiation than noncoding regions. Among the 389 markers, 158 could be mapped on the 12 linkage groups of the existing Q. robur genetic map. Outlier loci with large G(ST) values were distributed over 9 linkage groups. One cluster of three outlier loci was found within 0.51 cM; but significant autocorrelation of G(ST) was observed at distances <2 cM. The size and distribution of genomic regions involved in species divergence are discussed in reference to hitchhiking effects and disruptive selection.     

Comparison of earlywood vessel variables in the wood of Quercus robur L. and Quercus petraea (Mattuschka) Liebl. growing at the same site

The differences in the microscopic structure of wood based on the variables of earlywood vessel area and tree ring width were analysed in 6 trees of Pedunculate oak (Quercus robur L.) and 6 trees of Sessile oak (Quercus petraea (Mattuschka) Liebl.) in the same forest stand at a site in the Vizovice Highland (Czech Republic). The aim of this paper was to assess any differences between the two oak species when grown in the same location. Also the effect of tree-ring width and the effect of age on earlywood vessel variables were analysed. Mean values of tree-ring widths were not different between species. Earlywood vessel area chronologies were synchronized well between species. The mean values of earlywood vessel area (average vessel area, average vessel area of the first row of vessels, area of the largest vessel) showed a significant difference. Finally, we can conclude that there were differences in vessel features between these oak species.     

Identification of triterpene saponins in Quercus robur L. and Q. petraea Liebl. Heartwood by LC-ESI/MS and NMR

A detailed structural study of the major acidic triterpene saponins from European oak heartwood, Quercus robur L. and Q. petraea Liebl., revealed the presence of 2 alpha,3 beta,19 alpha-trihydroxyolean-12-ene-24,28-dioic acid (1), 2 alpha,3 beta,19 alpha-23-tetrahydroxyolean-12-ene-24,28-dioic acid (2), and their corresponding glycosides, 28-beta-D-glucopyranosyl-2 alpha,3 beta,19 alpha-trihydroxyolean-12-ene-24,28-dioic acid (3), and 28-beta-D-glucopyranosyl-2 alpha,3 beta,19 alpha,23-tetrahydroxyolean-12-ene-24,28-dioic acid (4). Compounds were isolated and purified by successive flash chromatography and semi-preparative HPLC, and structurally determined by NMR and LC-ESI/MS in the negative ion mode. Compounds 2 and 4 have been identified for the first time in Quercus species and are new compounds.     

Geographic structure of chloroplast DNA polymorphisms in European oaks

Chloroplast DNA polymorphisms have been detected by the conventional Southern-blotting hybridization method in four species of European oaks (Quercus petraea, Q. robur, Q. pubescens and Q. pyrenaica). Three polymorphisms, shared by at least three of these species, can be scored directly in ethidium bromidestained gels and were used in a broad survey of the level of differentiation of the oak species and of their pattern of genetic structure in western Europe. The highly significant geographic variation and the high genetic differentiation (Gst=0.895, S_Gst=0.025) indicate a low level of cytoplasmic gene flow. We conclude that cytoplasmic genomes are well suited for the reconstruction of past migrational routes of such a complex of species.     

Coincidence of small-scale spatial discontinuities in leaf morphology and nuclear microsatellite variation of Quercus petraea and Q. robur in a mixed forest

BACKGROUND AND AIMS: The taxon complex comprising Quercus petraea and Q. robur shows distinct morphologies and ecological preferences, but mostly low differentiation in various types of molecular markers at a broad spatial range. Local, spatially explicit analyses may reveal patterns induced by microevolutionary processes operating mainly over short distances. However, no attempts have been made to date to explore the potential of spatial analyses combining morphological and genetic data of these oaks. METHODS: A mixed oak stand was studied to elucidate the small-scale population genetic structure. All adult individuals were classified and putative hybrids were identified using multivariate discrimination analysis of leaf morphological characters. Likewise, all trees were genotyped with five nuclear microsatellites, and a Bayesian assignment method was applied based on maximum likelihood of multilocus genotypes for taxon and putative hybrid classification. KEY RESULTS: Multivariate analyses of leaf morphological data recognized two groups with few individuals as putative hybrids. These groups were significantly differentiated at the five microsatellites, and genetic taxon assignment coincided well with morphological classification. Furthermore, most putative hybrids were assigned to the taxon found in their spatial neighbourhood. When grouping trees into clusters according to their spatial positions, these clusters were clearly dominated by one taxon. Discontinuities in morphological and genetic distance matrices among these clusters showed high congruence. CONCLUSIONS: The spatial-genetic analyses and the available literature led to the assumption that reproductive barriers, assortative mating, limited seed dispersal and microsite-induced selection in favour of the locally adapted taxon at the juvenile stage may reinforce taxon-specific spatial aggregation that fosters species separation. Thus, the results tend to support the hypothesis that Q. petraea and Q. robur are distinct taxa which share a recent common ancestry. Occasional hybrids are rarely found in adults owing to selection during establishment of juveniles.     

Comparison of Isoprene Emission, Intercellular Isoprene Concentration and Photosynthetic Performance in Water-Limited Oak (Quercus pubescens Willd. and Quercus robur L.) Saplings

Abstract: The influence of prolonged water limitation on leaf gas exchange, isoprene emission, isoprene synthase activities and intercellular isoprene concentrations was investigated under standard conditions (30 °C leaf temperature and 1000 μmol photons m^-2 s^-1 PPFD) in greenhouse experiments with five-year-old pubescent oak ( Quercus pubescens Willd.) and four-year-old pedunculate oak ( Quercus robur L.) saplings. Net assimilation rates proved to be highly sensitive to moderate drought in both oak species, and were virtually zero at water potentials (Ψ_pd) below - 1.3 MPa in Q. robur and below - 2.5 MPa in Q. pubescens . The response of stomatal conductance to water stress was slightly less distinct. Isoprene emission was much more resistant to drought and declined significantly only at Ψ_pd below - 2 MPa in Q. robur and below - 3.5 MPa in Q. pubescens . Even during the most severe water stress, isoprene emission of drought-stressed saplings was still approximately one-third of the control in Q. robur and one-fifth in Q. pubescens . Isoprene synthase activities were virtually unaffected by drought stress. Re-watering led to partial recovery of leaf gas exchange and isoprene emission. Intercellular isoprene concentrations were remarkably enhanced in water-limited saplings of both oak species during the first half of the respective drought periods with maximum mean values up to ca. 16 μl l^-1 isoprene for Q. pubescens and ca. 11 μl l^-1 isoprene for pedunculate oak, supporting the hypothesis that isoprene serves as a short-term thermoprotective agent in isoprene-emitting plant species.     

European and Mediterranean oaks (Quercus L.; Fagaceae): SEM characterization of the micromorphology of the abaxial leaf surface

This paper examines micromorphological characteristics (trichomes, waxes, stomata) of the abaxial leaf surface of European and Mediterranean oaks. Observations were performed In-scanning electron microscopy, and the purpose of the investigation was to ascertain whether it is possible to distinguish the different species taxonomically using these features. Several species, as well as some subspecific entities and taxa considered doubtful, were taken into consideration. Subgenera appear to be well-defined: e.g. the subgenus Quercus is characterized by waxes arranged in vertical scales, the subgenera Cenis and Sclerophyllodris by smooth waxes. They differ because the former has markedly elliptical stomata, while the latter two have roundish stomata. The different species are also fairly easy to distinguish, and this fact confirms the taxonomical validity of the parameters we have taken into consideration. In some cases subspecific entities and doublful species do not diflcr at all from the related species, but in others they present such marked variations that it is difficult even to classify them with certainty. The most critical taxonomic groups (i.e. those which include a number of specific and subspecific entities often too difficult to distinguish from each other) are the series Quercus robur – Quercus petraea,– Quercus pubescens and the Quercus faginea,– Quercus Insitanica Quercus canariensis group.