Ecological and morphological characterisation, and phylogeny of Endomyxa: poorly known but widespread and diverse protozoan parasites and omnivores

Endomyxa are a very poorly known but large and diverse group of organisms in the protozoan phylum Cercozoa. Endomyxa includes the commercially important plasmodiophorid plant parasites, and haplosporidia and relatives, which include parasites of a wide range of invertebrates, most famously MSX disease of oysters. The evolutionary relationships of both of these groups were for a long time unknown (plasmodiophorids were considered fungi for a long time), but good ribosomal DNA trees show that they are related to each other (a relationship reinforced by morphological synapomorphies) and several lines of molecular evidence show that they belong within the eukaryotic supergroup Rhizaria, specifically as a subphylum within Cercozoa, but separately from the so-called 'core' Cercozoa. More recently, free-living relatives of these parasites have been found through culturing/cell isolation and rDNA sequencing. These include the large testate marine filose amoeba Gromia, and large reticulose, naked amoebae: the bacterivorous Filoreta and the predatory Arachnula and Platyreta. Such organisms represent very distinct and poorly studied protozoan morphotypes whose ecological roles are almost completely unknown. Other research has revealed more endomyxan parasites, for example the spot prawn parasite, and Paradinium, a parasite of copepods. Culture-independent environmental rDNA libraries show that there are a large diversity of other endomyxan clades and lineages which remain uncharacterised - the only information we have for them is the provenance of the samples in which the sequences were detected. The environmental libraries also show that there is very strong ecological structuring - many endomyxan clades have so far only been found in quite specific habitats, for example deep-sea samples, anaerobic marine, anaerobic freshwater, or phylloplane communities. These patterns suggest high levels of ecological specialization, perhaps involving interactions with other organisms. This project aims to identify as many of these novel lineages and clades as possible, whether they are free-living, symbiotic, or parasitic, using a combination of intensive selective culturing using a diversity of food sources and culture conditions informed by the results of the environmental libraries, and fluorescent in situ hybridization (FISH) using fluorescent probes that will specifically detect chosen uncharacterized lineages. FISH will also show the relative abundance of endomyxan lineages in different habitats, and whether they are associated with, or are found inside, other eukaryote cells, and if so, which organisms they co-occur with (and are therefore perhaps parasites of). Multiple and diverse SSU rDNA environmental libraries will be constructed, using a set of overlapping PCR primers covering all Endomyxa, to show much more clearly and realistically than previously possible the true diversity of Endomyxa, and their ecological nature by analyzing the sequence data in the context of other biotic and abiotic variables co-measured at each sampling site. The libraries will be made using both rDNA and crDNA reverse transcribed from rRNA: the latter is generally taken as a surrogate for the level of activity of the cells (more ribosomes will be present in more active cells), rather than just presence/absence and biomass as indicated by rDNA. This approach will provide more informative about the ecological characteristics than rDNA libraries alone. The environmental sequences will be used to improve phylogenetic reconstructions of Endomyxa and their relationships to the rest of Rhizaria both by increasing taxon sampling of this part of the tree, and by providing sequence data that can be used to obtain LSU rDNA sequences for representative genotypes directly from environmental DNA samples. Where cultures are available, other genes can be targeted by PCR, to be combined with existing data in multigene phylogenies.