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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Representational difference analysis reveals genomic differences between Q. robur and Q. suber: implications for the study of genome evolution in the genus Quercus

Very similar genome sizes, similar karyotypes and heterochromatin organisation, and identical number/position of ribosomal loci characterise the common oak (Q. robur) and the cork oak (Q. suber), two distantly related oak species. Representational Difference Analysis (RDA) was used to subtract the genome of Q. suber from the genome of Q. robur in order to search for genome differentiation. A library of 400 clones (bearing RDA fragments) representing genome differences between the two species was obtained. Seven Q. robur-specific DNA sequences were analysed with respect to their molecular and chromosome organisation. All belong to the dispersed repetitive component of the genome, as revealed by Southern hybridisation and in situ hybridisation. They are present in the Q. robur genome in between 100 and 700 copies, and are distributed along the length of almost all chromosomes. A search for homologies between RDA fragments and sequences in Genbank revealed similarities of all RDA fragments with known retrotransposons. The RDA fragments were also tested for their presence/absence in the genomes of six additional oak species belonging to different phylogenetic groups, in order to examine the evolutionary dynamics of these DNA sequences.     

Distribution of genomic regions differentiating oak species assessed by QTL detection

Pedunculate oak and sessile oak are two sympatric interfertile species that exhibit leaf morphological differences. We aimed to detect quantitative trait loci (QTLs) of these traits in order to locate genomic regions involved in species differentiation. A total of 15 leaf morphological traits were assessed in a mixed forest stand composed of Quercus petraea and Q. robur and in a full-sib pedigree of Q. robur. The progeny of the full-sib family were vegetatively propagated in two successive experiments comprising 174 and 216 sibs, and assessments were made on two leaves collected on each of the 1080 and 1530 cuttings corresponding to the two experiments. Traits that exhibited strong species differences in the mixed stand tended also to have higher repeatability values in the mapping population, thus indicating higher genetic control. A genetic map was constructed for QTL detection. Composite interval mapping with the one QTL model was used for QTL detection. From one to three QTLs were detected for 13 traits. In-depth analysis of the QTLs, controlling the five morphological traits that exhibited the highest interspecific differences in the mixed stand, indicated that they were distributed on six linkage groups, with two clusters comprising QTLs of at least two discriminant traits. These results were reinforced when error 1 for QTL detection was set at 5% at the chromosome level, as up to nine clusters could be identified. In conclusion, traits involved in interspecific differentiation of oaks are under polygenic control and widespread in clusters across the genome.     

Presence of triploids among oak species

BACKGROUND AND AIMS: Polyploids, organisms that have multiple sets of chromosomes, are common in certain plant and animal taxa. However, there are only a few reports of intraspecific ploidy variation within the genus Quercus. The aim of the study was to investigate the suspected ploidy level of two oaks that have unusual microsatellite banding patterns. METHODS: Polyploidy was investigated by using microsatellite analysis, stomata length measurements and nuclear DNA content estimation by flow cytometry. KEY RESULTS: Each putative triploid tree has patterns of microsatellite variation unexpected for diploid genomes, with up to three alleles at some loci, significantly longer stomata and 1.5 times more DNA per nucleus compared with diploids. CONCLUSIONS: To our knowledge, this report contains the first evidence for triploidy in Q. petraea and confirmation of this phenomenon in Q. robur. Regardless of the positive or negative aspects of the presence of triploid oaks in forest stands, it is of value to be able to screen for them. This study demonstrates that nuclear microsatellites and estimation of DNA content by flow cytometry can readily be used for this purpose.     

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.     

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.     

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 .     

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.     

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.     

Native oak chloroplasts reveal an ancient divide across Europe

Glacial refugia and postglacial migration are major factors responsible for the present patterns of genetic variation we see in natural populations. Traditionally postglacial history has been inferred from fossil data, but new molecular techniques permit historical information to be gleaned from present populations. The chloroplast tRNA^Leu1 intron contains regions which have been highly conserved over a billion years of chloroplast evolution. Surprisingly, in one of these regions which has remained invariant for all photosynthetic organisms so far studied, we have found intraspecific site polymorphism. This polymorphism occurs in two European oaks, Quercus robur and Q. petraea , indicating hybridisation and introgression between them. Two distinct chloroplast types occur and are distributed geographically as eastern and western forms suggesting that these oaks are each derived from at least two separate glacial refugia.