Background: Analysis of interspecific gene flow is crucial for the understanding of speciation processes and maintenance of species integrity. Oaks (genus Quercus, Fagaceae) are among the model species for the study of hybridization. Natural co-occurrence of four closely related oak species is a very rare case in the temperate forests of Europe. We used both morphological characters and genetic markers to characterize hybridization in a natural community situated in west-central Romania and which consists of Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto, respectively.

BackgroundFew studies address the issue of hybridization in a more than two-species context. The species-rich Quercus complex is one of the systems which can offer such an opportunity. To investigate the contemporary pattern of hybridization we sampled and genotyped 320 offspring from a natural mixed forest comprising four species of the European white oak complex: Quercus robur, Q. petraea, Q. pubescens, and Q. frainetto.ResultsA total of 165 offspring were assigned unambiguously to one of the pollen donors within the study plot. The minimum amount of effective pollen originating from outside the plot varied markedly among the seed parents, ranging from 0.18 to 0.87. The majority of the successful matings (64.1%) occurred between conspecific individuals indicating the existence of reproductive barriers between oak species. However, the isolation was not complete since we found strong evidence for both first-generation (8.4%) and later-generation hybrids (27.5%). Only two out of eight seed parents, belonging to Q. petraea and Q. robur, showed a high propensity to hybridize with Q. pubescens and Q. petraea, respectively. Significant structure of the effective pollen pools (Φpt = 0.069, P = 0.01) was detected in our sample. However, no support was found for the isolation by distance hypothesis. The proportion of hybrids was much higher (79%) in the seed generation when compared to the adult tree generation.ConclusionFirst-generation hybrids were observed only between three out of six possible species combinations. Hybrids between one pair of species preferred to mate with one of their parental species. The observation of first and later-generation hybrids in higher frequency in acorns than in adults might be explained by selection against hybrid genotypes, the history of this uneven-aged forest or past introgression between species.

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.