Use of flow cytometry and metabarcoding approaches to investigate the presence and abundance of the rare Glaucophyta in nature
University of New Brunswick
Eukaryotic cells acquired their photosynthetic machinery via primary endosymbiosis with cyanobacteria ~1 billion years ago. Various evidence suggests that the Archaeplastida lineage (rhodophytes, rhodelphids, picozoans, viridiplants and glaucophytes) descended from those first photosynthetic eukaryotes. However, the single origin of the Archaeplastida nucleo-cytoplasm host is still contentious and one of the main approaches for investigating that evolutionary scenario has been the limited data available for the rare Glaucophyta. The central aim of my dissertation is to establish reliable methods to detect, identify and isolate glaucophyte cells, or their genetic material, from the environment. Glaucophyte cells are characterized by a distinctive green-blue color due to the combination of blue phycobiliproteins (allophycocyanin and C-phycocyanin) with chlorophyll a. I used this set of photopigments in a cytometric investigation of glaucophyte autofluorescence (Chapter 2) to establish that this diagnostic feature can be used to distinguish glaucophyte cells in complex cell mixtures and isolate them to establish subcultures utilizing Fluorescence Activated Cell Sorting. The methodology presented provides a novel protocol to investigate glaucophytes, but also identified important challenges due to the low abundance of glaucophytes in nature. To overcome the challenges associated with such low abundances, it was then important to explore culture-independent methods, such as metabarcoding, which has become routine in surveys of microbial presence, abundance and diversity. In Chapter 3 of my thesis, I present a DNA metabarcoding survey of four different freshwater environments of southeast New Brunswick (Canada) to investigate microbial eukaryotic diversity, with the central goal of detecting glaucophytes. I detected members of the genus Glaucocystis in two of the collection sites at relatively high abundances when compared to the rest of the photosynthetic eukaryotic community. The presence of glaucophytes in local collection sites, together with some reports in the literature, laid the foundation for the work of Chapter 4, which includes the analysis of novel environmental samples and publicly available metabarcoding datasets. The aim of my last thesis chapter was to corroborate existing glaucophyte records and to present novel findings using a homogeneous bioinformatic workflow for reliably comparing the diverse datasets currently available. The two methodologies explored in my work provide a solid foundation for further investigations into the Glaucophyta and, more broadly, into any other rare microbial taxon.