Zygospore development of Spirogyra (Charophyta) investigated by serial block-face scanning electron microscopy and 3D reconstructions

Sexual reproduction of Zygnematophyceae by conjugation is a less investigated topic due to the difficulties of the induction of this process and zygospore ripening under laboratory conditions. For this study, we collected field sampled zygospores of Spirogyra mirabilis and three additional Spirogyra strains in Austria and Greece. Serial block-face scanning electron microscopy was performed on high pressure frozen and freeze substituted zygospores and 3D reconstructions were generated, allowing a comprehensive insight into the process of zygospore maturation, involving storage compound and organelle rearrangements. Chloroplasts are drastically changed, while young stages contain both parental chloroplasts, the male chloroplasts are aborted and reorganised as 'secondary vacuoles' which initially contain plastoglobules and remnants of thylakoid membranes. The originally large pyrenoids and the volume of starch granules is significantly reduced during maturation (young: 8 +/- 5 mu m(3), mature: 0.2 +/- 0.2 mu m(3)). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 +/- 18 mu m(3), mature: 0.1 +/- 0.2 and 0.5 +/- 0.9 mu m(3)). Only in S. mirabilis the LD volume increases (34 +/- 29 mu m(3)), occupying similar to 50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 mu m(3). The initially thin zygospore cell wall (similar to 0.5 mu m endospore, similar to 0.8 mu m exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (similar to 1.4 mu m) in Spirogyra sp. from Greece. The exo- and endospore show cellulose microfibrils in a helicoidal pattern. In the denser endospore, pitch angles of the microfibril layers were calculated: similar to 18 +/- 3 degrees in S. mirabilis, similar to 20 +/- 3 degrees in Spirogyra sp. from Austria and similar to 38 +/- 8 degrees in Spirogyra sp. from Greece. Overall this study gives new insights into Spirogyra sp. zygospore development, crucial for survival during dry periods and dispersal of this genus.