Fig. 1: A wide variety of protists inhabit anaerobic environments such as:
A) Salt Marshes
B) Mangroves
C) Termites.
|
Over the last decade, phylogenetic reconstructions of small subunit ribosomal DNA (ssu rDNA) sequences have provided a framework for understanding the course of early eukaryotic evolution. In these phylogenies, certain amitochondriate flagellate groups, notably diplomonads and parabasalids, consistently branch off before any of the mitchondria-bearing lineages. This finding suggested that mitochondria were acquired comparatively late in eukaryotic evolution, and that eukaryotes emerged in a low-oxygen environment, perhaps prior to the widespead oxygenation of Earth.
Currently, our knowledge of protistan diversity, particularly in the deep portion of the tree, is biased towards parasites of medical importance. Historical neglect of the free-living taxa makes it probable that have yet to sample much of the diversity of potentially deep-branching lineages. Access to these organisms will enhance our understanding of the deep phylogenetic structure of the eukaryotic tree and improve our inferences regarding the early history of the biosphere. Our approach is to isolate protists from unusual habitats including anaerobic sediments from mangroves, saltmarshes, and deep ocean vents and seeps that might be refuges for evolutionary relicts. These taxa are then subjected to detailed ultrastructural study in the laboratory of David J. Patterson. To determine the phylogenetic affinities of these organisms, we obtain tubulin (alpha and beta), elongation factor 1alpha (EF-1a) and ssu rDNA genes from the isolated anaerobic taxa and construct molecular phylogenies.
|
Methods: Anaerobic and aerobic protists are isolated from a variety of environments such as mangroves (Fig. 1A), saltmarshes (Fig. 1B), freshwater bogs, mudflats, freshwater, marine sediments or animal hosts (Fig. 1C) and cultured using media that meet their metabolic requirements. Once cultures are established, ultrastructural studies can be performed and nucleic acids can be isolated. Genes of interest are then amplified using degenerate primers developed by our laboratory that target highly-conserved regions of both protein and RNA-encoding genes. Amplification products are cloned and then sequenced using LI-COR automated sequencing technology. The resulting gene or inferred amino acid sequences entered into gene alignments. Phylogenetic relationships are inferred from the molecular and phenotypic data using parsimony, distance, and maximum likelihood methods implemented in the programs: PAUP*4.0, PHYLIP and PROTML.
|
