Assessment of flagellate diversity at deep-sea hydrothermal vents using the combined approach of culture-dependent and culture-independent methods

Abstract

Eighteen strains of flagellated protists representing 9 species were isolated and cultured from four deep-sea hydrothermal vents in the Eastern Pacific Ocean: Juan de Fuca Ridge, Guaymas Basin, and both 21°N and 9°N on the East Pacific Rise (EPR). The hydrothermal vent flagellates belonged to six different taxonomic orders: the Ancyromonadida, Bicosoecida, Cercomonadida, Choanoflagellida, Chrysomonadida, and Kinetoplastida. Molecular and ultrastructural evidence point to one of the isolates, Ancyromonas, as a plausible candidate for the closest relative to the common ancestor of Metazoans, Fungi, and Choanoflagellates (the Opisthokonta). Using l8S rDNA sequences from most of the major eukaryotic lineages, maximum likelihood, minimum evolution and maximum parsimony analyses yielded congruent phylogenies supporting this hypothesis. Deep-sea vent samples were both cultured to select for kinetoplastid flagellates and analyzed without culturing by denaturing gradient gel electrophoresis (DGGE) using PCR primers specific to the kinetoplastid clade. By comparing these two different methods of analysis, my goal was to decrease the biases and/or errors inherent in either method alone and to improve our ability to assess flagellate diversity and distribution in samples from remote vent environments. PCR and DGGE were used to specifically isolate and amplify target DNA's from all cultured kinetoplastid species in matching vent samples, thus corroborating the findings of culturing. Molecular methods had the additional ability to detect species presence where culturing did not, thereby providing a better indication of the distribution of these species. Many of the vent isolates were ubiquitous members of marine, freshwater, and terrestrial ecosystems worldwide, suggesting a global distribution of these flagellate species. This discovery advanced the hypothesis that ubiquity in distribution patterns among heterotrophic flagellates implies high tolerance and/or adaptability to a wide range of environmental conditions. Experiments under vent conditions of high pressure and high concentrations of metals and sulfide showed that some of these species are very tolerant to extreme environmental conditions.