Marine:freshwater transitions in choanoflagellates

Choanoflagellates are ubiquitous and one of the most important groups of heterotrophic pico- and nanoplankton. They are the closest relatives to metazoans and are considered a very valuable group for the study of the early evolution, in animals, of some key life traits as important as sexual reproduction or multicellularity. The phylogeny of freshwater choanoflagellates remains poorly understood, with only one major group containing most of the diversity of freshwater cultured species. Conversely, previous studies have estimated a high hidden biodiversity amongst freshwater choanoflagellates that have led to the emergence of clades composed solely of environmental sequences. Such disparities between the known and estimated diversity of freshwater choanoflagellates pose a major barrier towards the understanding of the evolutionary history of marine:freshwater transitions in choanoflagellates. By a combination of exhaustive phylogenetic analysis, ancestral sequence reconstruction, and phylogenetic placement of environmental sequences, the present study has sought to investigate these discrepancies, in order to unravel the evolutionary history of marine:freshwater transitions in choanoflagellates. Furthermore, the present study has focused on the assessment of the tolerance to salinity changes in several species of choanoflagellates, in order to identify candidates for future studies focused on the molecular mechanisms of adaptation to varying salinities. Additionally the present study has involved extensive efforts towards the isolation of new species of choanoflagellates species from rare freshwater habitats, more specifically acidic and hyperalkaline environments. As a result of the analysis carried out, the present study has revealed that three independent colonisations of freshwater environments have occurred throughout the evolutionary history of Clade 2 choanoflagellates. Similarly, the results have identified three major hotspots of hidden diversity within choanoflagellates. Such hotspots are located at basal positions of Acanthoecida, Craspedida, and within Clade 2 salpingoecids. The estimated hidden diversity within Clade 2 salpingoecids is of marine origin and indicates that, within this group, freshwater colonisations events might be much more frequent than previously thought. The estimated hidden diversity of the other two hotspots is of both marine and freshwater origin indicating that multiple lineages of both marine and freshwater choanoflagellates remain unknown and that marine:freshwater transitions have occurred independently several times throughout the evolutionary history of choanoflagellates. The results from the autoecology of Microstomoeca roanoka revealed that this species is a prime candidate for the study of the molecular mechanisms of adaptation to varying salinities in choanoflagellates. Lastly, the present study contains the description of four morphospecies of choanoflagellates, two from hyperalkaline and two from acidic environments.