RESUMEN
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 µm³, mature: 0.2 ± 0.2 µm³). In contrast, lipid droplets (LDs) increase significantly in number upon zygospore maturation, while simultaneously getting smaller (young: 21 ± 18 µm³, mature: 0.1 ± 0.2 and 0.5 ± 0.9 µm³). Only in S. mirabilis the LD volume increases (34 ± 29 µm³), occupying ~50% of the zygospore volume. Mature zygospores contain barite crystals as confirmed by Raman spectroscopy with a size of 0.02 - 0.05 µm³. The initially thin zygospore cell wall (~0.5 µm endospore, ~0.8 µm exospore) increases in thickness and develops a distinct, electron dense mesospore, which has a reticulate appearance (~1.4 µm) 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: ~18 ± 3° in S. mirabilis, ~20 ± 3° in Spirogyra sp. from Austria and ~38 ± 8° 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.
RESUMEN
Understanding the desiccation and freezing tolerance of bryophyte spores is vital to explain how plants conquered land and current species distribution patterns and help to develop efficient ex situ conservation methods. However, knowledge of these traits is scarce. We investigated tolerance to drying (at 15% relative humidity [RH] for two weeks) and freezing (1 h exposure to liquid nitrogen) on the spores of 12 bryophyte species (23 accessions) from the UK. The presence of storage lipids and their thermal fingerprint, and the levels of unfrozen water content, were determined by differential scanning calorimetry (DSC). The presence of chlorophyll in dry spores was detected by fluorescence microscopy. All species and accessions tested tolerated the drying and freezing levels studied. DSC suggested that 4.1−29.3% of the dry mass is storage lipids, with crystallization and melting temperatures peaking at around −30 °C. Unfrozen water content was determined <0.147 g H2O g−1 dry weight (DW). Most of the spores investigated showed the presence of chlorophyll in the cytoplasm by red autofluorescence. Bryophyte spores can be stored dry at low temperatures, such as orthodox seeds, supporting the creation of bryophyte spore banks. However, the presence of storage lipids and chlorophyll in the cytoplasm may reduce spore longevity during conventional storage at −20 °C. Alternatively, cryogenic spore storage is possible.