Format: ORAL

Enrico Boccato, Fabio Candotto Carniel, Mauro Tretiach

Department of Life Sciences, University of Trieste, Trieste, Italy

The aero-terrestrial green microalgae of the genus Trebouxia Puymaly are the most frequent photobionts of lichens, but our comprehension of their sexual life is very limited, because in the lichen symbiosis it is typically suppressed by the fungal partner. In this study, innovative techniques were applied to characterise the formation and destiny of zoospores in four axenically cultured Trebouxia species(T. decolorans, T. vagua, T. gelatinosa, and T. angustilobata),selected to represent three of the four primary phylogenetic clades(A, A, I, and S, respectively)recognized within the genus. Frequent zoosporogenesis was achieved by increasing the concentration of glucose and peptone in the growth solid medium. By light transmitted and scanning electron microscopy(SEM)on samples treated with theCO2 critical point dryer, we identified two distinct zoospore morphotypes, conserved among the major clades of the genus: spherical to sub-spherical vs. broadly ellipsoidal to ovoid. By observing zoospores stained with OsO4 at the SEM in backscattered electrons mode, we could easily identify lipid bodies, as confirmed by the detection of Os by energy dispersive X-ray spectrometry, whose presence varied among species. These lipid bodies, which serve as energy sources in motile gametes of other green algae, led us to hypothesise that the heteromorphic zoospores of Trebouxia could represent true gametes. Sexual reproduction was corroborated in T. decolorans by confocal observations, through the assessment of the DNA content at cell population level, thus providing the first insights into the ploidy of this organism. Finally, cells with four flagella, which might be interpreted as the first direct evidence of a kariogamic event, were documented by light and SEM microscopy in populations of zoospores isolated by cytofluorimetry.The role of heteromorphic zoospores in the life cycle of Trebouxia and the implications of sexual reproduction in the photobionts of lichens will be thoroughlydiscussed.