RESEARCH PAPER Natural hybridisation between Quercus petraea (Matt.) Liebl. and Quercus pubescens Willd. within an Italian stand as revealed by microsatellite fingerprinting D. Salvini 1,2 , P. Bruschi 3 , S. Fineschi 1 , P. Grossoni 3 , E. D. Kjær 2 & G. G. Vendramin 4 1 Istituto per la Protezione delle Piante, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Firenze, Italy 2 Skov og Landskab, København Universitet, Hørsholm, Denmark 3 Dipartimento di Biologia Vegetale, Universita ` di Firenze, Firenze, Italy 4 Istituto di Genetica Vegetale, Consiglio Nazionale delle Ricerche, Sesto Fiorentino, Firenze, Italy INTRODUCTION Interspecific gene flow is frequently reported in the genus Quercus (Gonzalez-Rodriguez et al. 2004; Tovar-Sanchez & Oyama 2004; Valbuena-Caraban ˜a et al. 2005). How- ever, interfertile oak species often seem to remain distinct, even within areas of sympatry (Craft et al. 2002) and, despite their genomic similarity, both their morphology and ecophysiology can be highly differentiated. Hence, oaks have created interest in the debate over the role of hybridisation in plant evolution and diversification (Petit 2004). Recently, the use of DNA markers combined with mor- phological identification has provided more objective quantification of hybridisation in oaks. Three main types of evidence have been presented: (i) wide sharing of chlo- roplast DNA haplotypes in areas of sympatry (Dumolin- Lapegue et al. 1999; Belahbib et al. 2001); (ii) limited interspecific genetic differentiation at neutral loci (Bruschi et al. 2000; Gonzalez-Rodriguez et al. 2004; Tovar-San- chez & Oyama 2004; Valbuena-Caraban ˜a et al. 2005); and (iii) hybridisation revealed by parentage analysis (Dow & Ashley 1998; Streiff et al. 1999; Valbuena-Caraban ˜a et al. 2005). Keywords Hybridisation; microsatellite; paternity analysis; Quercus petraea; Quercus pubescens. Correspondence S. Fineschi, CNR – IPP, via Madonna del Piano 10, I-50019 Sesto Fiorentino – FI, Italy. E-mail: s.fineschi@ipp.cnr.it Editor F. Salamini Received: 26 June 2008; Accepted: 10 September 2008 doi:10.1111/j.1438-8677.2008.00158.x ABSTRACT Interspecific gene flow is frequently reported in the genus Quercus. How- ever, 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 subsam- ple of such trees were collected and analysed using molecular microsatellite markers. A low interspecific genetic differentiation and a high level of inter- specific 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 fi 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 pol- linations). Intermediate ‘mother trees’ were pollinated in similar propor- tions 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. Plant Biology ISSN 1435-8603 758 Plant Biology 11 (2009) 758–765 ª 2008 German Botanical Society and The Royal Botanical Society of the Netherlands