Antonio Marco Nardella’s research while affiliated with University of Naples Federico II and other places

A comparative investigation was made of floral scent variation in the closely related, food-rewarding Anacamptis coriophora and the food-deceptive Anacamptis morio in order to identify patterns of variability of odour compounds in the two species and their role in pollinator attraction/avoidance learning. Scent was collected from plants in natural populations and samples were analysed via quantitative gas chromatography and mass spectrometry. Combined gas chromatography and electroantennographic detection was used to identify compounds that are detected by the pollinators. Experimental reduction of scent variability was performed in the field with plots of A. morio plants supplemented with a uniform amount of anisaldehyde. Both orchid species emitted complex odour bouquets. In A. coriophora the two main benzenoid compounds, hydroquinone dimethyl ether (1,4-dimethoxybenzene) and anisaldehyde (methoxybenzaldehyde), triggered electrophysiological responses in olfactory neurons of honey-bee and bumble-bee workers. The scent of A. morio, however, was too weak to elicit any electrophysiological responses. The overall variation in scent was significantly lower in the rewarding A. coriophora than in the deceptive A. morio, suggesting pollinator avoidance-learning selecting for high variation in the deceptive species. A. morio flowers supplemented with non-variable scent in plot experiments, however, did not show significantly reduced pollination success. Whereas in the rewarding A. coriophora stabilizing selection imposed by floral constancy of the pollinators may reduce scent variability, in the deceptive A. morio the emitted scent seems to be too weak to be detected by pollinators and thus its high variability may result from relaxed selection on this floral trait.

Natural hybridization between plant species often occurs in disturbed habitats and it is generally considered a threat for rare and endangered species. A different situation occurs in Mediterranean food deceptive orchids, where hybridization is a common phenomenon, as a natural consequence of their unspecific pollination system.Here, we present molecular and ecological evidence from a hybrid zone between Orchis mascula and O. pauciflora in order to address the consequences of hybridization on local orchid evolution and to assess conservation priorities for hybrids and hybrid zones in orchid conservation programs.We find that, although hybrids among the two target taxa are formed relatively frequently, hybrid zones often consist of F1 individuals, whereas backcrosses and later generation hybrids appear to be either rare or absent, presumably as a consequence of post-zygotic reproductive barriers acting in these food-deceptive orchids. Experimental evidence further indicates that hybrids are less fit than the parental species in attracting pollinators and that parental taxa exhibit higher or similar fitness in sympatry compared to allopatric populations. Since introgression is not frequent as indicated by molecular analyses, fitness and phenotypic trait differences observed among sympatric and allopatric populations of the parental taxa may be a consequence of pollinator-mediated selection.Our experimental data confirm that hybridization is a natural phenomenon in food-deceptive orchids and that it does not pose a threat to their survival. Furthermore, sympatric zones provide the stage for evolutionary processes in orchids, and this peculiarity should be taken into account when devising orchid conservation strategies.

Pollinator specificity has traditionally been considered the main reproductive isolation mechanism in orchids. Among Mediterranean orchids, however, many species attract and deceive pollinators by mimicking nectar-rewarding plants. To test the extent to which deceptive orchid species share pollinators, we collected and identified hemipollinaria-carrying insects, and used ribosomal sequences to identify the orchid species from which hemipollinaria were removed. We found that social and solitary bees, and also flies, carried hemipollinaria belonging to nine orchid species with different degrees of specialization. In particular, Anacamptis morio, Dactylorhiza romana and Orchis mascula used a large set of pollinator species, whereas others such as Orchis quadripunctata seemed to be pollinated by one pollinator species only. Out of the insects with hemipollinaria, 19% were found to carry hemipollinaria from more than one orchid species, indicating that sympatric food-deceptive orchids can share pollinators. This sharing was apparent even among orchid sister-species, thus revealing an effective overlap in pollinator sets among closely related species. These results suggest varying degrees of pollinator specificity in these orchids, and indicate that pollinator specificity cannot always act as the main isolation mechanism in food-deceptive temperate orchids.