• Phylogenetic signals
• orbicules' morphology
• Gymnosperm
• free orbicules
• daily aerobiological samples
• Cupressaceae
• confocal-light microscopes
• asthma attacks
• Angiosperm
• ancestral state reconstructions
• pollen grains
• pollinosis
• SEM
• Taxaceae

Orbicules are sub- or pauci-micronic particles associated with pollen grains and tapetum cells. In Angiosperms, they are common in the ANITA grade and monocots, but are absent in the Asteraceae and the majority of Lamiaceae, revealing an evolutionary trend from presence to absence along the phylogenetic tree. However, for 20 orders of angiosperms no data on the presence-absence of orbicules is available.
Recent studies have highlighted the importance of orbicules in terms of the systematic value or as vectors of allergens that trigger pollinosis. Previous research has postulated the presence of orbicules in the atmosphere, however the first report of their direct observations was published as a result of the present doctorate project. Their size, shape and density are useful systematic characters in the tribal characterization within families of Gentianales. While orbicules have indeed been reported for most families and genera of allergologic importance, their usefulness in systematics remains little explored.
Accordingly, my reasearch is divided into three sections. In the first one, I provided the first direct evidence that orbicules are released into the atmosphere by detecting them in aerobiological samples.
I observed pollen sacs, pollen grains and orbicules of nine species of Cupressaceae using scanning electron microscope (SEM).
Under SEM, I measured orbicule size (0.494 to 0.777 µm) and detected unknown nanometric particles (130 to 200 nm). Under the light microscope, aerobiological samples showed clusters of stained dots surrounding the pollen grains. Under the confocal microscope, the same clusters were resolved into submicron particles with the same autofluorescence as the pollen grains. These features enabled me to identify them as orbicules.
I believe that my findings help to explain the onset of pollinosis and allergic asthma related to Cupressaceae pollen grains in many countries, even before pollen grains are actually detected or after they are no longer observed in aerobiological monitoring samples.
In the second section my study aims: a) to discover the presence/absence of orbicules in species of allergological interest hitherto overlooked regarding these particles; b) to propose standard descriptors for orbicule morphology, and c) to link the features of orbicules to a systematic framework.
I looked for orbicules in pollen sacs and pollen grains of 23 species of 17 genera in Asteraceae, Betulaceae, Fagaceae, Oleaceae, Poaceae, Plantaginaceae and Polygonaceae. Wherever applicable, I measured categorical and continuous traits of orbicules, included them in a matrix and then subjected it to non-metric multidimensional scaling (NMDS) as ordination method to assess the usefulness of orbicule traits as systematic characters.
I detected and described orbicules on the tapetum and on the pollen exine in 19 target species and observed unknown nanoparticles that might a) explain the process of deposition of sporopollenin during orbicule development, and b) contribute to the onset of pollinosis – along with orbicules - before and after the dispersion of pollen grains into the air. Finally, within the restricted sample examined, the NMDS technique applied to selected orbicule traits allowed to resolve the dataset at species level.
In the third section, I focused on the gaps of knowledge about presence-absence of orbicules and investigated 34 species from 29 different genera and 14 orders, for which no data was available: Acorales, Arecales, Buxales, Celastrales, Crossosomatales, Dilleniales, Escalloniales, Garryales, Gunnerales, Paracryphiales, Santalales, Trochodendrales and Zygophyllales. I photographed pollen sacs and pollen grains with a SEM, then described and measured selected orbicule traits of the target species. I then calculated the phylogenetic signals for continuous and discrete variables and reconstructed their ancestral states for the whole set of 57 species investigated during this doctorate project, belonging to 18 orders.
Orbicule traits are supported both by a strong phylogenetic signal and by a significant evolution trait value.
The evolution rate for absence-presence of orbicules shows an adaptive radiation, while phylogenetic signals for sizes and density of the orbicules indicate evolutionary convergences.
Furthermore, I detected an evolutionary trend towards a reduction in size and density of orbicules, that might have led to the absence of the orbicules in the most derived clades.