Zoospore diversity and sexual reproduction in the lichen-forming genus Trebouxia: From neglected evidence to new facts.

Trebouxia is one of the most frequent genera of green microalgae that occur as photobionts in lichens. However, its life cycle is still poorly understood. The contradictory information about the flagellate cells impairs our knowledge of possible sexual reproduction. The aim of this study was to investigate the behaviour and fate of flagellate cells in four Trebouxia species, to better understand their role in the life cycle. Axenic cultures of Trebouxia were grown under controlled conditions. The cell cycle of flagellate cells was observed using a novel technique for real-time monitoring with light microscopy, validated in more detail by scanning electron microscopy (SEM). Additionally, a molecular approach was used to investigate genomic evidence of sexual reproduction. The zoospores had two significantly different morphotypes, elongated and subspherical, suggesting that this dimorphism should be recognised in future species descriptions. Fusions of elongated gametes were observed in three species, with formation and development of a zygote documented in one case. SEM images provided further evidence of plasmogamic events in unprecedented detail. Molecular analyses confirmed the presence of key meiotic genes in eight genomes and one transcriptome of Trebouxia, providing further strong evidence of sexual reproduction in this genus. This study provides a new method to monitor the fate of flagellate cells over time which allowed demonstration of the presence of two types of flagellate cell: zoospores with two well-defined morphologies which are involved in asexual reproduction, and gametes involved in sexual reproduction.