Genetic variation of oaks (<Emphasis Type="Italic">Quercus</Emphasis> spp.) in Switzerland. 3. Lack of impact of postglacial recolonization history on nuclear gene loci

Quercus petraea, Quercus pubescens and Quercus robur are closely related and interfertile white oaks native to Switzerland. The three species are known to share identical cpDNA haplotypes, which are indicative of the postglacial recolonization history of populations. Only two haplotypes are common in Switzerland. We compared variation of cpDNA and of isozymes in 28 oak populations from Switzerland in order to assess the impact of the postglacial population history on current genetic structures of nuclear controlled isozyme gene loci. Species delineation was based on Principal Component Analysis of leaf morphological traits. The species status of populations was reflected at isozyme gene loci, but differentiation between populations with different cpDNA haplotypes and hence different recolonization history was very low at enzyme gene loci for all species. Thus, glacial and postglacial population history was not reflected at nuclear gene loci on the temporal and spatial scale covered by the present study. Extensive gene flow through pollen among populations is likely to have blurred a previously existing genetic differentiation at biparentally inherited gene loci that possibly evolved in the different glacial refugia of the above mentioned cpDNA haplotypes.     

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.     

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.     

Fusarium eumartii Growth in Resistant and Susceptible Oak Species

Fusarium eumartii is a fungus associated with declining Quercus robur , in which it is found in the vessels. The response of oak species to infection is known to vary: Q. robur is susceptible , but Quercus cerris and Quercus pubescens are resistant. An experiment was carried out in 1996 and repeated in 1997, to examine how F. eumartii colonization differed in oak species that were susceptible or resistant to the fungus by counting the number of vessels with mycelium at various distances from the inoculation site in infected seedlings and by determining the amount of viable fungus in infected tissue. Infected vessels with mycelium were counted on sections (10  μ m thick) cut at 0, 2, 4, 6, 8 and 10 cm from the inoculation site on 1-year-old inoculated seedlings as well as on sections cut every 2 cm to the seedling tip. The amount of viable fungus was determined by counting the colony forming units (CFUs) in stem segments from the same seedlings. Quercus robur seedlings had the greatest number of infected vessels and the greatest number of CFUs. Forty days after inoculation, the extent of vertical fungal spread was 28.12 cm in Q. robur , 3.15 cm in Q. cerris and 3.00 cm in Q. pubescens . The greatest number of CFUs was found in Q. robur at day 5 after inoculation. Analysis of variance confirmed the results.     

AFLP markers demonstrate local genetic differentiation between two indigenous oak species [ Quercus robur L. and Quercus petraea (Matt.) Liebl.] in Flemish populations

The nuclear genetic variation within and between four sessile ( Q. petraea) and six pedunculate ( Q. robur) autochthonous Flemish oak populations was investigated with AFLP markers. One sessile and one pedunculate oak population were additionally screened for detailed leaf characteristics using an image analysis system. Principal coordinate analysis on the AFLP data classified the oaks in two main groups, according to their taxonomic status. No species-specific AFLP markers were found using four primer combinations, but marker frequency differences up to 71% were recorded between both species. Analysis of the genetic structure showed that the divergence between species, as observed by ordination, was significant. Both species revealed similar diversity levels. A smaller though significant differentiation was also revealed for both species among populations within species. Molecular and morphology based approaches showed a high degree of consistency. Screening of 60 AFLP primer combinations using a bulking strategy did not allow identifying species-specific markers, which supports the conclusions reached in previous studies. The distribution of genetic variability at the species and at the population level is discussed.     

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.     

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.     

Potential host plants of Corythucha arcuata (Het., Tingidae) in Europe: a laboratory study

Abstract:  The oak lace bug Corythucha arcuata (Say) (Het., Tingidae), native to North America, was found in Europe on Quercus robur L. and other oaks in the spring of 2000. The potential host plant range of this species in Europe and its development time were investigated in a laboratory study. An assay was performed on leaf cuts of different plant species. On the deciduous European oaks ( Q. robur , Quercus pubescens Willd, Quercus petraea (Mattuschka) Liebl., Quercus cerris L.), as well as Rubus ulmifolius Schott. and Rubus idaeus L., most of the lace bugs (>50%) reached the adult stage; on Castanea sativa Mill., Rubus caesius L. and Rosa canina L., a reduced number of individuals (<25%) reached the adult stage. No nymphs survived on Quercus rubra L. (mentioned in literature as a host plant), on the evergreen oaks Quercus suber L. and Quercus ilex L., on Malus domestica Borkh. and four tested maple species. On plant species where the lace bug reached the adult stage, the development time varied from 13 to 27 days. On European deciduous oak species, the development time was longer on leaves taken in late summer (September) than on those of late spring (June); on the contrary, such differences were not observed on Rubus species, and Castanea sativa .     

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.     

Morphological and molecular diversity among Italian populations of Quercus petraea (Fagaceae)

Quercus petraea (sessile oak) has a scattered distribution in southern and central Italy. The objective of this work was to evaluate the level and distribution of diversity in five Italian populations of Q. petraea by using morphological markers and hypervariable molecular markers such as microsatellites. Forty-eight morphological traits and six nuclear and three plastid loci were scored for each population. Evidence for differentiation in both sets of traits was found, but patterns of differentiation of morphological traits did not coincide with microsatellite differentiation. Morphological variation was correlated with ecological conditions at the site of origin. Analysis of molecular variance revealed significant genetic variation among populations (P < 0.001), both at the nuclear and plastid levels. There was a slight, but significant, correlation between nuclear genetic distance and geographic distance. The relatively high genetic diversity in the populations analysed indicates that the maintenance of their evolutionary potential is possible if population sizes are maintained or increased. Low levels of haplotype diversity found within the small southernmost population (Piano Costantino) indicates that genetic erosion may increase the extinction risk for this population.     

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.     

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.     

The clonal structure of Quercus geminata revealed by conserved microsatellite loci

The scrub oak communities of the southeastern USA may have existed at their present locations for thousands of years. These oaks form suckers, and excavations of root systems suggest that clones may occupy very large areas. Resolution of the clonal nature of scrub oaks is important both to manage the tracts of this ecosystem that remain, and in conducting long-term ecological studies, where the study area must substantially exceed the area occupied by any single clone. Microsatellites were used to determine the genetic diversity of a dominant oak species within a 2-ha long-term experimental site on Merritt Island at the Kennedy Space Center. This area contains a long-term study of the effects of elevated CO2 on the ecosystem. Conservation of seven microsatellite loci, previously identified in the sessile oak, Quercus petraea, was tested in two Florida scrub oak species, Q. geminata and Q. myrtifolia. Sequence analysis revealed that all seven microsatellite loci were conserved in Q. geminata and five loci were conserved in Q. myrtifolia. Six microsatellite loci were polymorphic in Q. geminata and these were subsequently used to investigate the clonal structure of the Q. geminata population. Twenty-one unique combinations of microsatellites, or haplotypes, occurred only once, whereas the remaining 26 individuals belonged to a total of seven different haplotypes. Trees with identical haplotypes were in close proximity, supporting the interpretation that they were clones. The results showed that there is significant genetic diversity within the 2-ha experimental site. Microsatellites provided a powerful and noninvasive tool for distinguishing individual genotypes and determining an adequate area for long-term ecosystem studies.     

Three divergent rDNA clusters predate the species divergence in Quercus petraea (Matt.) Liebl. and Quercus robur L

Quercus petraea and Quercus robur are two closely related oak species that frequently hybridize. We sequenced 70 clones containing the 5.8S and ITS2 regions of ribosomal DNA (rDNA) from these two species and did not detect a species-specific difference. Surprisingly, three divergent (up to 12.6%) rDNA families were identified in both species, indicating that they predate the speciation event. Despite a large between-rDNA-families divergence, rDNA sequences were very similar within families, suggesting ongoing concerted evolution. Expression analysis, relative-rate tests, and mutation spectrum analyses indicated that only a single rDNA family is functional. We propose that past hybridization events, combined with nucleolar dominance, were the evolutionary processes underlying the contemporary rDNA variability in Q. petraea and Q. robur.     

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.     

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.     

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.     

Pure species in a continuum of genetic and morphological variation: sympatric oaks at the edge of their range

Background and Aims Studies on oaks (Quercus spp.) have often been hampered by taxonomic confusion, a situation further compounded by the occurrence of extensive interspecific hybridization. In the present study, a combination of genetic and morphological analyses was used to examine sympatric populations of Q. petraea and Q. robur at the north-western edge of their ranges in Northern Ireland, since it had previously been suggested that hybridization could facilitate the apparent rapid, long-distance dispersal of oaks following the glaciations.Methods Samples were collected from 24 sites across Northern Ireland that had been previously designated as ancient or semi-natural woodland. Genotypes were obtained from a total of 950 trees using 12 nuclear microsatellite loci, and admixture coefficients were calculated based on a Bayesian clustering approach. Individuals were also classified as Q. petraea, Q. robur or hybrids based on two objective morphometric characters shown previously to delineate pure individuals effectively. Genetically ‘pure’ individuals of both species, as defined by the Bayesian clustering, were also genotyped for five chloroplast microsatellites.Key Results Genetic and morphological analyses both indicated the presence of pure individuals of both species, as well as a continuum of intermediates. There was a good agreement between the molecular and morphological classification, with a generally clear separation between pure individuals.Conclusions Despite millennia of hybridization and introgression, genetically and morphologically pure individuals of both Q. petraea and Q. robur can be found at the edge of their range, where both species occur sympatrically. The high proportion of individuals exhibiting introgression compared with previous studies may reflect the historical role of hybridization in facilitating dispersal following the glaciations. This is further supported by the significantly higher chloroplast diversity in Q. robur compared with Q. petraea.     

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.     

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.