Two microalgal species, Trebouxia jamesii and Trebouxia sp. TR9, were detected as the main photobionts coexisting in the thalli of the lichen Ramalina farinacea. Trebouxia sp. TR9 emerged as a new taxon in lichen symbioses and was successfully isolated and propagated in in vitro culture and thoroughly investigated. Several years of research have confirmed the taxon Trebouxia sp. TR9 to be a model/reference organism for studying mycobiont–photobiont association patterns in lichen symbioses. Trebouxia sp. TR9 is the first symbiotic, lichen-forming microalga for which an exhaustive characterization of cellular ultrastructure, physiological traits, genetic and genomic diversity is available. The cellular ultrastructure was studied by light, electron and confocal microscopy; physiological traits were studied as responses to different abiotic stresses. The genetic diversity was previously analyzed at both the nuclear and organelle levels by using chloroplast, mitochondrial, and nuclear genome data, and a multiplicity of phylogenetic analyses were carried out to study its intraspecific diversity at a biogeographical level and its specificity association patterns with the mycobiont. Here, Trebouxia sp. TR9 is formally described by applying an integrative taxonomic approach and is presented to science as Trebouxia lynnae, in honor of Lynn Margulis, who was the primary modern proponent for the significance of symbiosis in evolution. The complete set of analyses that were carried out for its characterization is provided.

The lichenized green microalga Trebouxia lynnae Barreno has been recently described and is considered a model organism for studying lichen chlorobionts. Its cellular ultrastructure has already been studied in detail by light, electron, and confocal microscopy, and its nuclear, chloroplast and mitochondrial genomes have been sequenced and annotated. Here, we investigated in detail the ultrastructure of in vitro grown cultures of T. lynnae observed by Low Temperature Scanning Electron Microscopy (LTSEM) applying a protocol with minimum intervention over the biological samples. This methodology allowed for the discovery of ultrastructural features previously unseen in Trebouxiophyceae microalgae. In addition, original Transmission Electron Microscopy (TEM) images of T. lynnae were reinterpreted based on the new information provided by LTSEM. The nucleolar vacuole, dictyosomes, and endoplasmic reticulum were investigated and reported for the first time in T. lynnae and most likely in other Trebouxia lineages.

Lichen phycobiomes have recently emerged as a source of biodiversity and new species of microalgae. Although in the genus Diplosphaera free-living microalgae are common, numerous strains belonging to this genus have frequently been recognized or isolated from lichen thalli. In this study, a comprehensive analysis of the strain Diplosphaera sp. ASUV135, isolated from a lichen thallus, has been carried out using an integrative taxonomic approach. The SSU and nuclear-encoding ITS rDNA, as well as the chloroplast rbcL gene, were sequenced and analyzed to ascertain its taxonomic position and phylogenetic relationships within the genus Diplosphaera. This strain was also analyzed by light, confocal and transmission microscopy for morphological and ultrastructural characterization. The phenotypic plasticity in this strain was also confirmed by changes in its morphology under different growth conditions, as well as those of modulated Chlorophyll a fluorescence emissions, to understand its photosynthetic functioning. Our results pointed out that this strain represents a new taxon within the genus Diplosphaera (Prasiola group), described here as Diplosphaera elongate sp. nova. This study also provides tools for future research on organisms with high phenotypic plasticity by using molecular, morphological, ultrastructural and physiological approaches.

Lichens are a mutualistic symbiosis between a fungus and one or more photosynthetic partners. They are photosynthetically active during desiccation down to relative water contents (RWCs) as low as 30% (on dry mass). Experimental evidence suggests that during desiccation, the photobionts have a higher hydration level than the surrounding fungal pseudo-tissues. Explosive cavitation events in the hyphae might cause water movements towards the photobionts. This hypothesis was tested in two foliose lichens by measurements of ultrasonic acoustic emissions (UAEs), a method commonly used in vascular plants but never in lichens, and by measurements of PSII efficiency, water potential, and RWC. Thallus structural changes were characterized by low-temperature scanning electron microscopy. The thalli were silent between 380% and 30% RWCs, when explosive cavitation events should cause movements of liquid water. Nevertheless, the thalli emitted UAEs at ~5% RWC. Accordingly, the medullary hyphae were partially shrunken at ~15% RWC, whereas they were completely shrunken at <5% RWC. These results do not support the hypothesis of hyphal cavitation and suggest that the UAEs originate from structural changes at hyphal level. The shrinking of hyphae is proposed as an adaptation to avoid cell damage at very low RWCs.