Molecular Ecology (2007) 16, 2855–2866 doi: 10.1111/j.1365-294X.2007.03240.x © 2007 The Authors Journal compilation © 2007 Blackwell Publishing Ltd Blackwell Publishing Ltd The strength of reproductive isolation in two hybridizing food-deceptive orchid species MARIA DOMENICA MOCCIA,* ALEX WIDMER † and SALVATORE COZZOLINO * *Dipartimento delle Scienze Biologiche, Università degli Studi di Napoli Federico II, Via Foria, 223, I-80139 Naples, Italy, †ETH Zurich, Plant Ecological Genetics, Institute of Integrative Biology, Universitätstrasse 16, 8092 Zurich, Switzerland Abstract Reproductive isolation is of fundamental importance for maintaining species boundaries in sympatry. In orchids, the wide variety of pollination systems and highly diverse floral traits have traditionally suggested a prominent role for pollinator isolation, and thus for prezygotic isolation, as an effective barrier to gene flow among species. Here, we examined the nature of reproductive isolation between Anacamptis morio and Anacamptis papiliona- cea, two sister species of Mediterranean food-deceptive orchids, in two natural hybrid zones. Comparative analyses of the two hybrid zones that are located on soils with volcanic origin and have different and well-dated ages consistently revealed that all hybrid indi- viduals were morphologically and genetically intermediate between the parental species, but had strongly reduced fitness. Molecular analyses based on nuclear ITS1 and (amplified fragment length polymorphism) AFLP markers clearly showed that all examined hybrids were F 1 hybrids, and that no introgression occurred between parental species. The mater- nally inherited plastid DNA markers indicated that hybridization between A. morio and A. papilionacea was bidirectional, as confirmed by the molecular analysis of seed families. The genetic architecture of the two hybrid zones suggests that the two parental species easily and frequently hybridize in sympatry as a consequence of partial pollinator overlap but that strong postzygotic barriers reduce hybrid fitness and prevent gene introgression. These results corroborate that chromosomal divergence is instrumental for reproductive isolation between these food-deceptive orchids and suggest that hybridization is of limited importance for their diversification. Keywords: Anacamptis morio, Anacamptis papilionacea, hybrid sterility, hybrid zones, karyotype divergence, reproductive barriers Received 26 August 2006; revision accepted 13 November 2006 Introduction Species integrity between closely related sympatric species depends on the strength of reproductive isolation. In flowering plants with overlapping phenology, reproductive isolation may be achieved by specific pollinators, post– pollination barriers or a combination of both mechanisms (Grant 1981). In orchids, the extraordinary floral diversity indicates the importance of orchid–pollinator associations for orchid evolution and orchid pollination biology is regarded as a driving force in orchid diversification and speciation (Cozzolino & Widmer 2005a). In particular, the high specificity of orchid–pollinator associations has been proposed as the main reproductive isolation mechanism among orchid species and consequently, a prominent role has been allocated to prezygotic reproductive isolation mechanisms in this plant lineage (Dodson & Gillespie 1967; Gill 1989). Contrary to expectations, however, high levels of natural hybridization have been well documented among species and genera of terrestrial, Mediterranean orchids (Willing & Willing 1977, 1985 and references therein), which contrasts sharply with the view of highly specialized orchid–pollinator associations. These obser- vations have promoted the idea that orchid pollination is not always species-specific, in particular in the Mediter- ranean area (Cozzolino & Widmer 2005b). Many of these hybridizing orchids display a food- deceptive pollination system: they have large, conspicuous flowers that offer no reward and either attract and deceive Correspondence: Salvatore Cozzolino, Fax: +39081450165; E-mail: cozzolin@unina.it