Defining DNA-Based Operational Taxonomic Units for Microbial-Eukaryote Ecology

Abstract

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.Ecological studies of aquatic microbial eukaryotes require identification and enumeration of organisms with extremely wide taxonomic diversity. The assemblages are typically dominated by phototrophic and heterotrophic protists (microalgae and protozoans), but microscopic metazoans belonging to a variety of animal phyla can also contribute significantly. Identification of protists in environmental samples is particularly difficult because most species have been defined morphologically (41, 84). Protistan identification involves a wide variety of procedures for collection, preservation, specimen preparation, and examination (34, 84), as well as many different types of taxonomic expertise. Very few studies have attempted to identify and enumerate all protistan taxa because of these complexities, which makes it difficult to evaluate ecological studies of protistan diversity, community structure, and biogeochemical function.The growing database of DNA sequence information for a wide spectrum of microbial eukaryotes offers the possibility for greatly improving the existing tools for studying the phylogeny and ecology of these organisms. Much of the initial impetus for the acquisition ...

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“…This is lower than the cutoff value for most environmental DNA studies (e.g. Caron et al 2009). Indeed, previous studies have explored different cut-off levels ranging from 90 to 100 % (Stoeck et al 2007;Edgcomb et al 2011;Orsi et al 2011;Hadziavdic et al 2014), which is necessary for any new primer set used to amplify a fragment of the 18S rRNA gene.…”

“…Nevertheless, there are, as yet, no universally accepted guidelines to this cut-off value, which ranges from 90 to 99 % (e.g. Stoeck et al 2007;Caron et al 2009;Marande et al 2009;Schnetzer et al 2011;Edgcomb et al 2011;Orsi et al 2011). In our study, we used a modified approach with a highly stringent clustering cut-off value of 99 %.…”

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