Notes: We compared 16S-like ribosomal RNA (rRNA) coding regions of samples of the solitary spumellarian radiolarian Thalassicolla nucleata collected from the Sargasso Sea and the Pacific Ocean. Sequences derived from these locations showed variability in both length and base-pair composition. This level of sequence variability is similar to the degree of variability reported in the literature for species- or even genus-level distinctions. Explanations for our results include multiple alleles for the rRNA gene, or the existence of multiple species of Thalassicolla that are morphologically indistinguishable. The seven existing descriptions of Thalassicolla species, including T. nucleata, are discussed in view of these molecular findings and with reference to our current understanding of the physiology and life cycle of the spumellarian radiolaria.

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: . Thirty clones derived from twenty isolates of heterotrophic nanoflagellates originating from a variety of marine and freshwater environments were examined by restriction fragment length polymorphism analysis of small subunit ribosomal RNA genes amplified by the polymerase chain reaction (riboprinting). The data were compared with light and electron microscopical identification of the isolates. On morphological criteria, sixteen of the thirty clones belonged to the genus Paraphysomonas De Saedeleer, seven to the genus Spumella Cienkowski, four to the genus Pteridomonas Penard and three to the genus Cafeteria Fenchel and Patterson. Among these taxa, eleven ribotypes were detected by analysis with the restriction enzymes Hinf I, Hae III, Sau 3A I, and Msp I. Differentiation of nanoflagellate taxa by the riboprinting method supported taxonomic classification based on morphology at the generic and species level. The utility of the method for discriminating the ‘naked’ flagellates and for confirming the identity of polymorphic forms among species of Paraphysomonas is demonstrated.

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: . Modern genetic and immunological techniques have become important tools for assessing protistan species diversity for both the identification and quantification of specific taxa in natural microbial communities. Although these methods are still gaining use among ecologists, the new approaches have already had a significant impact on our understanding of protistan diversity and biogeography. For example, genetic studies of environmental samples have uncovered many protistan phylotypes that do not match the DNA sequences of any cultured organisms, and whose morphological identities are unknown at the present time. Additionally, rapid and sensitive methods for detecting and enumerating taxa of special importance (e.g. bloom-forming algae, parasitic protists) have enabled much more detailed distributional and experimental studies than have been possible using traditional methods. Nevertheless, while the application of molecular approaches has advanced some aspects of aquatic protistan ecology, significant issues still thwart the widespread adoption of these approaches. These issues include the highly technical nature of some of the molecular methods, the reconciliation of morphology-based and sequence-based species identifications, and the species concept itself.

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.

Notes: 1. The occurrence and grazing activity of mixotrophic (phagotrophic) algae in three icecovered freshwater lakes of different trophic status were examined (oligotrophic Lake Giles, mesotrophic Lake Lacawac, eutrophic Lake Waynewood), Microbial population densities were low (4.1–7.2 × 105 bacteria ml−1 and 1.2–2.4 × 103 nanoplanktonic protists ml−1). All three nanoplankton communities were dominated by chloroplast-bearing forms (60–96%).2. Mixotrophs formed up to 48% of the phototrophic nanoplankton in Lake Lacawac and were responsible for up to ∼90% of the observed uptake of bacteria-sized particles. The abundance of mixotrophic algae in Lakes Giles and Waynewood were extremely low (3 and 2% of the phototrophic algae, respectively), and heterotrophs dominated nanoplankton bacterivory.3. The overall impact of nanoplankton feeding activity on the bacterial assemblage was low under the ice in Lakes Giles and Waynewood. Removal rates of bacteria based on our particle uptake experiments were 1.0 and 4.0% of the bacterial standing stock day−1 in these lakes, respectively. Removal rates were higher in Lake Lacawac and ranged from 4.9 to 11% of the bacterial standing stock day−1 on 2 successive sampling days.