Defining DNA-based operational taxonomic units for microbial-eukaryote ecology.

DNA sequence information has increasingly been used in ecological research on microbial eukaryotes. Sequence-based approaches have included studies of the total diversity of selected ecosystems, studies of the autecology of ecologically relevant species, and identification and enumeration of species of interest for human health. It is still uncommon, however, to delineate protistan species based on their genetic signatures. The reluctance to assign species-level designations based on DNA sequences is in part a consequence of the limited amount of sequence information presently available for many free-living microbial eukaryotes and in part a consequence of the problematic nature of and debate surrounding the microbial species concept. Despite the difficulties inherent in assigning species names to DNA sequences, there is a growing need to attach meaning to the burgeoning amount of sequence information entering the literature, and there is a growing desire to apply this information in ecological studies. We describe a computer-based tool that assigns DNA sequences from environmental databases to operational taxonomic units at approximately species-level distinctions. This approach provides a practical method for ecological studies of microbial eukaryotes (primarily protists) by enabling semiautomated analysis of large numbers of samples spanning great taxonomic breadth. Derivation of the algorithm was based on an analysis of complete small-subunit (18S) rRNA gene sequences and partial gene sequences obtained from the GenBank database for morphologically described protistan species. The program was tested using environmental 18S rRNA data sets for two oceanic ecosystems. A total of 388 operational taxonomic units were observed for 2,207 sequences obtained from samples collected in the western North Atlantic and eastern North Pacific oceans.

Distinct protistan assemblages characterize the euphotic zone and deep sea (2500 m) of the western North Atlantic (Sargasso Sea and Gulf Stream).

Protistan diversity was characterized at three locations in the western North Atlantic (Sargasso Sea and Gulf Stream) by sequencing 18S rRNA genes in samples from euphotic (< or = 125 m) and bathypelagic depths (2500 m). A total of 923 partial-length protistan sequences were analysed, revealing 324 distinct operational taxonomic units (OTUs) determined by an automated OTU-calling program set to 95% sequence similarity. Most OTUs were comprised of only one or two sequences suggesting a large but rare pool of protistan diversity. Many OTUs from both depth strata were associated with recently described novel alveolate and stramenopile lineages while many OTUs from the bathypelagic were affiliated with Acantharea, Polycystinea and Euglenozoa and were not observed in euphotic zone libraries. Protistan assemblages from the euphotic zone and the deep sea were largely composed of distinct OTUs; only 28 of the 324 protistan OTUs were detected in both shallow and deep sea clone libraries. The diversity of protistan assemblages in the deep sea was distinctly lower than the diversity of euphotic zone assemblages. Protistan assemblages from the Gulf Stream were the most diverse for either depth strata. Overall, protistan assemblages from different stations but comparable depths were more similar than the assemblages from different depths at the same station. These data suggest that particular groups of protistan OTUs formed distinct 'shallow' and 'deep-sea' assemblages across widely spaced oceanic locales.

Protistan diversity estimates based on 18S rDNA from seawater incubations in the Western North Atlantic.

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.