F. Bellusci’s research while affiliated with University of Calabria and other places

Floral displays, influencing attractiveness to insects, increase the number of pollinator visits and the efficiency of each visit in terms of pollen exchange and thus affect the plant reproductive success. Here, we conducted an in situ manipulation experiment to investigate whether the floral modifications affect reproductive success in natural orchid populations of Serapias lingua and Serapias vomeracea. We estimated male and female reproductive success of three treatment groups, disassembly of floral tube, cutting of lip, and painting of the callus surface, in terms of pollinaria removed/deposited and fruit production. Results revealed that phenotypic modification had opposite effects on reproductive success of two examine species. Indeed, reproductive success was significantly increased by the detached of the petals and sepals, and decreased, due to callus painting and lip removal, in S. lingua. On the contrary, unmanipulated plants of S. vomeracea showed significantly higher value of pollinaria removed and deposited and fruit set than manipulated ones. The differences between S. lingua and S. vomeracea agree to the different pollination strategy of examined species. S. vomeracea shows shelter imitation strategy, and thus, the disassembly of tunnel-like corolla does not allow the insects to use the flower as a refuge, while S. lingua is a sexually deceptive orchid and therefore the opening of the flower made more visible callus (visible at a greater distance) increasing the pollinators attraction. This study provides evidence that pollinators were largely sensitive to the experimental modification of the flower phenotype, which is consistent with the presence of significant selection on individual floral characters. Our experimental investigations of the effects of variation in display on pollinator visitation provide insights into the evolution of floral morphology in orchid with shelter imitation strategy.

Reproductive success of plants may be affected by interactions with co-flowering species either negatively, through competition for pollinators, or positively, by means of a magnet species effect and floral mimicry. In this study, potential interactions between Iris tuberosa, a rewarding species, and Ophrys fusca, a sexually deceptive orchid, were explored in four populations in southern Italy. In each population plots showing different ratios of the examined species were arranged in the field, and in each plot the number of pollinators and fruit set were assessed. In addition, flower size and floral hydrocarbons produced by the two species were analysed. Morphological and scent data pointed out that flower size and aliphatic compounds did not differ significantly between the two species. Interestingly, both species shared tricosane and 11-nonacosene, electrophysiologically active compounds in the shared dominant pollinator Adrena. We have found that fruit production and number of pollinators in I. tuberosa varied significantly among plots, while percentage of capsules and number of pollinators of O. fusca captured showed no significant differences across plots. These results suggested, that the presence of O. fusca contributes differentially to pollinator attraction, and thus, to total reproductive success of I. tuberosa, according to a different ratio of aggregation. These findings suggest that I. tuberosa profits from the greater abundance of insects attracted by the presence of orchid specimens, and that a sexually deceptive orchid may be a magnet species in pollination strategy.

Background: Fragmentation of habitats by roads, railroads, fields, buildings and other human activities can affect population size, pollination success, sexual and asexual reproduction specially in plants showing pollinator limitation, such as Mediterranean orchids. In this study, we assessed pollen flow, selfing rates, vegetative reproduction and female reproductive success and their correlations with habitat characters in nine fragmented subpopulations of Serapias lingua. To improve understanding of population structure effects on plant biology, we examined genetic differentiation among populations, pollen flow, selfing rates and clonal reproduction using nuclear microsatellite markers. Results: Smaller populations showed a significant heterozygote deficit occurred at all five nuclear microsatellite loci, the coefficient of genetic differentiation among populations was 0.053 and pairwise FST was significantly correlated with the geographical distance between populations. Paternity analysis of seeds showed that most pollen flow occurred within a population and there was a positive correlation between percentage of received pollen and distance between populations. The fruit production rate varied between 5.10 % and 20.30 % and increased with increasing population size, while the percentage of viable seeds (78-85 %) did not differ significantly among populations. The extent of clonality together with the clonal and sexual reproductive strategies varied greatly among the nine populations and correlated with the habitats where they occur. The small, isolated populations tended to have high clonal diversity and low fruit production, whereas the large populations with little disturbance were prone to have reductions in clonal growth and increased sexual reproduction. Conclusions: We found that clonality offers an advantage in small and isolated populations of S. lingua, where clones may have a greater ability to persist than sexually reproducing individuals.

Orchidaceae is one of the most species-rich angiosperm families, and all orchids are fully dependent on fungi for their seed germination and their life cycle. The level of specificity of the association between orchid species and fungi can be related to the number of co-occurring orchid species. To investigate orchid mycorrhizal associations in adult-photosynthetic orchids, 16 Mediterranean orchid species belonging to 4 genera (Anacamptis, Ophrys, Orchis, and Serapias) at 11 different sites were subjected to DNA-based analysis. Eighteen operational taxonomic units representing two fungal families, Tulasnellaceae and Ceratobasidiaceae, were identified. All examined orchid species associated with different mycorrhizal fungi. Interestingly, there was a positive correlation between number of orchid species and number of mycorrhizal. Monospecific populations showed a lower number of fungi, while sympatric populations had a higher number of mycorrhizal fungi. Our results showed that Mediterranean orchid species associated with a higher number of mycorrhizal fungi confirming as photosynthetic orchids are typically generalists toward mycorrhizal fungi. Thus, photosynthetic orchids exhibit low specificity for fungal symbionts showing the potential for opportunistic associations with diverse fungi reducing competition for nutrient. We suggest that these characteristics could confer symbiotic assurance particularly in habitat with resource limitations or prone to stressful conditions.

Fragmentation of habitats by roads, railroads, fields, buildings and other human activities can affect population size, pollination success and fruit production, especially in plants showing pollinator limitation, such as Mediterranean orchids. In this study, we investigated the effect of human activity on the population dynamics and reproductive success of the orchid Serapias cordigera. Three anthropic and three natural populations were monitored over 14 years (1999–2012), classifying individuals into five stage classes and evaluating reproductive success. Population growth rates differed between anthropic and natural populations. Our results demonstrated that small anthropic populations have lower population viability compared with large natural populations. The proportion of flowering plants, the number of reproductive plants and the percentage of fruits were significantly lower in anthropic than in natural populations. This strong decline in fruit production in populations in urban areas may reflect lower pollination attraction and higher inbreeding in small than in natural populations. Calculation of extinction probabilities showed that the anthropic populations will drop below the survival threshold of 15–20 years. This study highlights that continued monitoring is needed to improve information on population viability and for appropriate conservation management. © 2014 The Linnean Society of London, Botanical Journal of the Linnean Society, 2014, 176, 408–420.

The frequency of hybrid formation in angiosperms depends on how and when heterospecific pollen is transferred to the stigma, and on the success of that heterospecific pollen at fertilising ovules. We applied pollen mixtures to stigmas to determine how pollen interactions affect siring success and the frequency of hybrid formation between two species of Mediterranean deceptive orchid. Plants of Orchis italica and O. anthropophora were pollinated with conspecific and heterospecific pollen (first conspecific pollen then heterospecific pollen and vice versa) and molecular analysis was used to check the paternity of the seeds produced. In this pair of Mediterranean orchids, competition between conspecific and heterospecific pollen functions as a post-pollination pre-zygotic barrier limiting the frequency of the formation of hybrids in nature. Flowers pollinated with heterospecific pollen can remain receptive for the arrival of conspecific pollen for a long time. There is always an advantage of conspecific pollen for fruit formation, whether it comes before or after heterospecific pollen, because it overtakes the heterospecific pollen. The conspecific pollen advantage exhibited in O. italica and O. anthropophora is likely to result from the reduced germination of heterospecific pollen or retarded growth of heterospecific pollen tubes in the stigma and ovary. Overall, the results indicate that our hybrid zone represents a phenomenon of little evolutionary consequence, and the conspecific pollen advantage maintains the genetic integrity of the parental species.

The nuclear ribosomal DNAwas used to identify the orchid mycorrhizal fungi found in roots of Orchis xbivonae and its parental species Orchis anthropophora and Orchis italica. Polymerase chain reaction products were sequenced and identified using the expanded database.We determined that closely related Tulasnellaceae are mycorrhizal in the three orchid taxa, suggesting that the mycorrhizal partner does not impair hybrid survival. This study demonstrates that O. xbivonae displays few differences in comparison with its two parental species in identity of its associated mycorrhizal fungi, it is a short- -term by-product of the hybridizing behavior of common pollinators, and thus it will not easily origin descendents with potential new genetic combinations and/or ecological preferences.

Orchid species of Mediterranean genus Serapias often live in sympatry, exhibit similar floral morphology, bloom in the same period and share the same pollinators. Previous studies on Serapias species have ascertained that reproductive isolation is based on pre-pollination barriers, that secretory cells and trichomes are typically distributed on the floral labellum and that flowers produce aliphatic compounds. In this study we compare the floral scent composition of four widespread, co-occurring Serapias species, namely Serapias lingua, Serapias parviflora, Serapias vomeracea and Serapias cordigera. Our goals are to assess if differences in floral scent may act as interspecific pre-pollination barriers and if these olfactory signals may be involved in the pollination strategy of Serapias. We find that all the selected species produce C20–C29 alkanes and alkenes and, in addition, have detected the presence in S. cordigera of large amounts of oleate and stearate ethyl ester. Our findings help to clarify that the sympatric Serapias species have slightly different floral scent signatures that may account for their relevant role as pre-pollination barriers. Therefore, the pollination strategy of Serapias relies not only on the tubular shape of their floral corolla but also on the production of olfactory signals that may lure potential pollinators and even assure a sufficient degree of pollinator fidelity.

Despite highly specialized pollination strategies, hybridization is a common phenomenon among Mediterranean deceptive orchids. Food-deceptive species sire a progeny of F1 unfertile plants, which work as a late post-zygotic barrier. Conversely, when pre-zygotic barriers of sexually deceptive (Ophrys) species are absent, the hybrids are fertile and an extensive introgression may occur. Here, we have performed molecular analysis and hand pollination treatments to characterize a hybrid zone of two food-deceptive species, O. mascula and O pauciflora. Hybrids (called O. × colemanii) have shown different amounts of parental nrDNA, strongly supporting that they are F2 and/or successive hybrid generations. Comparable high levels of reproductive success have been detected in natural conditions and in experimental crosses suggesting the absence of effective reproductive barriers either between hybrids, either between hybrids and parental species. In light of ecological and distributional features of O. × colemanii across its distribution range, we hypothesize that these populations have originated by secondary contact in the periglacial belt of Apennines. Moreover, the rare and localized O. pauciflora could benefit a genetic enrichment by hybridizing with a widespread related species. O. × colemanii is not a dead end population, but may have a role as potential reserve of adaptive variability and is an unusual stage along the speciation process.

A great variety of colours, shapes, scents, energy‐rich rewards and other floral traits form an integrated system that increases pollination efficiency or reduces pollen loss. Flowers lacking energetic rewards possess a more elaborate morphology to ensure co‐specific pollination. Despite the prevalence of deceptive species among Euro‐Mediterranean orchids, the study of their floral micromorphology is still in its infancy. In this work, we examine the floral micromorphology of four sympatric species of the widespread Mediterranean genus Serapias. Electron microscopy (scanning, transmission) was performed on flowers of four sympatric species of the deceptive genus Serapias, the widespread S. cordigera, S. lingua, S. parviflora and S. vomeracea. Each species has a characteristic combination of specialized, secretory epidermal cells and trichomes on the labellum and, to a lesser extent, the petals. Serapias lingua is highly specialized in that it produces olfactory signals and has two types of trichome with secretory apical cells, one of which is also present in S. parviflora. In addition to striated, conical secretory cells, S. cordigera and S. vomeracea also have characteristic, atypical secretory trichomes with bilobulate or dome‐like protuberances. The labellar micromorphology of S. lingua and S. parviflora is adapted for scent production, whereas S. vomeracea and S. cordigera possess trichomes that may also provide visual and/or tactile cues. Owing to the combined effect of the specific floral fragrance and labellar micromorphology in pollinator selection, the pollination ecology of Serapias spp. is even more specialized than previously thought. © 2012 The Linnean Society of London, Botanical Journal of the Linnean Society, 2012, 169, 714–724.