Coordinated ultrastructural and phylogenomic analyses shed light on the hidden phycobiont diversity of Trebouxia microalgae in Ramalina fraxinea.

The precise boundary delineations between taxa in symbiotic associations are very important for evolutionary and ecophysiological studies. Growing evidence indicates that in many cases, the use of either morphological characters or molecular markers results in diversity underestimation. In lichen symbioses, Trebouxia is the most common genus of lichen phycobionts, however, the diversity within this genus has been poorly studied and as such there is no clear species concept. This study constitutes a multifaceted approach incorporating aspects of ultrastructural characterization by TEM and phylogenomics to evaluate the morphological and genetic diversity of phycobionts within the sexually reproducing lichen Ramalina fraxinea in the context of Mediterranean and temperate populations. Results reveal an association with at least seven different Trebouxia lineages belonging to at least two species, T. decolorans and T. jamesii, and diverse combinations of such lineages coexisting within the same thallus depending on the analyzed sample. Some of these lineages are shared by several other non-related lichen taxa. Our findings indicate the existence of a highly diverse assemblage of Trebouxia algae associating with R. fraxinea and suggest a possible incipient speciation within T. decolorans rendering a number of lineages or even actual species. This study stresses the importance of coordinated ultrastructural and molecular analyses to improve estimates of diversity and reveal the coexistence of more than one Trebouxia species within the same thallus. It is also necessary to have clearer species delimitation criteria within the genus Trebouxia and microalgae in general.