Rare unclassified 16S rRNA operational taxonomic units from the uncharted Engaño Bay (Argentinean Patagonia).

Rare microbes make up most of the diversity of marine microbiomes, and recent works have highlighted their importance for microbial community dynamics and in fragmented habitats. Rare taxa have been infrequently studied in comparison with abundant groups, and rare unclassified sequences are common in culture-independent studies. Here, we describe a detailed analysis of nonclassifiable sequences from the Chubut river estuary at the Argentinean Patagonia. Standard taxonomic assignments of environmental 16S rRNA sequences resulted in about 13% unclassified operational taxonomic units (OTUs). The potential affiliations of these OTUs could be narrowed by mapping the classification software assignments on a phylogeny obtained directly from our environmental sequence data. Customized BLAST analyses were remarkably consistent with these phylogenetic assignments, especially when the unclassified OTUs were blasted against sequences from cultured and type microorganisms. In addition, our BLAST analyses revealed significant similarities between several unclassified OTUs and a plethora of unclassified sequences from around the world. Further phylogenetic comparisons with 6194 carefully selected reference sequences showed that these unclassified sequences may correspond to 5 unnamed groups, possibly encompassing ranks from subclass to family inside the Alphaproteobacteria, and to an unknown Gracilibacteria lineage. Overall, these results demonstrate the value of straight phylogenetic analysis, customized BLAST searches, and comparisons with sequences from type material, for the systematic study of rare unclassified sequences.