Notes: Abstract. Cloning/sequencing and fragment analysis of ribosomal RNA genes (rDNA) are becoming increasingly common methods for the identification of microbial taxa. Sequences of these genes provide many additional taxonomic characters for species that otherwise have few distinctive morphological features, or that require involved microscopy or laboratory culture and testing. These same approaches are now being applied with great success in ecological studies of natural communities of microorganisms. Extensive information on the composition of natural microbial assemblages is being amassed at a rapid pace through genetic analyses of environmental samples and comparison of the resulting genetic information with well-established (and rapidly growing) public databases. We examined microbial eukaryote diversity in a natural seawater sample from the coastal western North Atlantic Ocean using two molecular biological approaches: the cloning and sequencing of rRNA genes and by fragment analysis of these genes using the terminal restriction fragment length polymorphism (T-RFLP) method. A simple experiment was carried out to examine changes in the overall eukaryote (largely protistan) diversity and species composition (phylotype diversity) of a natural microbial assemblage when a seawater sample is placed in a container and incubated at ambient light and temperature for 72 h. Containment of the natural seawater sample resulted in relatively minor changes in the overall eukaryote diversity (species richness) obtained by either molecular method at three time points (time-zero, time-24 h, time-72 h). However, substantial changes in the dominance of particular eukaryote phylotypes took place between the three sampling times. Only 18% of the total number of phylotypes observed in the study were observed at all three time points, while 65% (108 of 165) phylotypes were observed only at a single time point (54 unique phylotypes initially, 37 more unique phylotypes at 24 h, and 17 more at 72 h). The results of this study indicate that a high diversity of protistan taxa existed in the original seawater sample at very low abundance, and thus were not observed in the initial characterization of community structure. Containment resulted in significant shifts in the dominance of these taxa, enabling the presence of previously unobserved phylotypes to be documented after 24 or 72 h of incubation.

Notes: Traditional microscope-based studies of protistan assemblages in the Ross Sea, Antarctica have contributed significantly to our understanding of the microbial biogeography and food web structure in this extreme cold-water environment. However, these investigations have neither been able to characterize the genetic diversity of the communities, nor have they necessarily determined the abundances or trophic contribution of the dominant organisms. Resolution of the latter issue is hindered by the fact that physiological studies are often conducted on opportunistic species that respond to enrichment culture rather than species truly representative of intact communities. Therefore, we sought to determine the genetic diversity of microbial eukaryotes in ice, water and slush samples from the Ross Sea, and establish both morphologic and physiologic links between enrichment cultures and the genetic data. Denaturing gradient gel electrophoresis and ribosomal clone library analyses indicate that the protistan communities are very diverse, but those present in similar habitats are more alike than those in different habitats at the same site. We have been able to identify several protists from our enrichment cultures as being genetically represented in the original samples. General studies of their physiology have been initiated and methods for determining their abundances are being developed.

Notes: Ice, slush, sediment and water samples were collected from the Ross Sea, Antarctica and enriched with a variety of inorganic and organic nutrients. All cultures were maintained at an ambient temperature of 1°C, and amoebae were observed to occur in the slush, sediment and water enrichments. Amoebae were isolated into clonal or monocultures, and their characterization was accomplished using a combination of molecular and morphological methods. Full-length 18S ribosomal DNA sequence data indicated that seven of the isolates represented four different amoebae of the Vexilliferidae and Vannellidae families. Acquisition of 18S ribosomal sequences from the parasomes of two amoebae further confirmed their identification as Neoparamoeba species. Light microscopy, fluorescence microscopy and TEM observations were accomplished and further support the molecular data. To determine whether these amoebae represented psychrophilic strains, their temperature tolerances were tested. Cultures were inoculated at 1°C and the temperature was raised 1°C/day (to a total of 5°), and then held at that temperature for 6 days. This pattern was repeated in 5°-increments, up to 20°C. The cultures were observed frequently for death and possible cyst formation. Three (2 types of Vannellids) out of seven amoeba cultures survived up to 20°C. Whereas, the two different Vexilliferids died at a range of temperatures between 10°C and 20°C. These data indicate that amoebae found in the Antarctic are varied in their physiological adaptation for growth at cold temperatures.