Dataset Information

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.

ABSTRACT: Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial 'rare biosphere'. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular methods relates to that revealed by classical morphology approaches is practically nonexistent. To address this issue, we used different approaches to assess the diversity of tintinnid ciliates, a species-rich group in which species can be easily distinguished morphologically. We studied two Mediterranean marine samples with different patterns of tintinnid diversity. We estimated tintinnid diversity in these samples employing morphological observations and both classical cloning and sequencing and pyrosequencing of two different markers, the 18S rDNA and the internal transcribed spacer (ITS) regions, applying a variety of computational approaches currently used to analyze pyrosequence reads. We found that both molecular approaches were efficient in detecting the tintinnid species observed by microscopy and revealed similar phylogenetic structures of the tintinnid community at the species level. However, depending on the method used to analyze the pyrosequencing results, we observed discrepancies with the morphology-based assessments up to several orders of magnitude. In several cases, the inferred number of operational taxonomic units (OTUs) largely exceeded the total number of tintinnid cells in the samples. Such inflation of the OTU numbers corresponded to 'rare biosphere' taxa, composed largely of artifacts. Our results suggest that a careful and rigorous analysis of pyrosequencing data sets, including data denoising and sequence clustering with well-adjusted parameters, is necessary to accurately describe microbial biodiversity using this molecular approach.

SUBMITTER: Bachy C

PROVIDER: S-EPMC3554406 | biostudies-literature | 2013 Feb

REPOSITORIES: biostudies-literature

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Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.

Bachy Charles C Dolan John R JR López-García Purificación P Deschamps Philippe P Moreira David D

The ISME journal 20121004 2

Deep-sequencing technologies are becoming nearly routine to describe microbial community composition in environmental samples. The 18S ribosomal DNA (rDNA) pyrosequencing has revealed a vast diversity of infrequent sequences, leading to the proposition of the existence of an extremely diverse microbial 'rare biosphere'. Although rare microbes no doubt exist, critical views suggest that many rare sequences may actually be artifacts. However, information about how diversity revealed by molecular m ...[more]

PMID: 23038176

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Project description:Ciliates represent central nodes in freshwater planktonic food webs, and many species show pronounced seasonality, with short-lived maxima of a few dominant taxa while many being rare or ephemeral. These observations are primarily based on morphospecies counting methods, which, however, have limitations concerning the amount and volume of samples that can be processed. For high sampling frequencies at large scales, high throughput sequencing (HTS) of freshwater ciliates seems to be a promising tool. However, several studies reported large discrepancy between species abundance determinations by molecular compared to morphological means. Therefore, we compared ciliate DNA metabarcodes (V9 regions of the 18S rRNA gene) with morphospecies counts for a 3-year study (Lake Zurich, Switzerland; biweekly sampling, n = 74). In addition, we isolated, cultivated and sequenced the 18S rRNA gene of twelve selected ciliate species that served as seeds for HTS analyses. This workflow allowed for a detailed comparison of V9 data with microscopic analyses by quantitative protargol staining (QPS). The dynamics of V9 read abundances over the seasonal cycle corroborated well with morphospecies population patterns. Annual successions of rare and ephemeral species were more adequately characterized by V9 reads than by QPS. However, numbers of species specific sequence reads only partly reflected rank orders seen by counts. In contrast, biomass-based assemblage compositions showed higher similarity to V9 read numbers, probably indicating a relation between cell sizes and numbers / sizes of macronuclei (or 18S rRNA operons). Full-length 18S rRNA sequences of ciliates assigned to certain morphospecies are urgently needed for barcoding approaches as planktonic taxa are still poorly represented in public databases and the interpretation of HTS data depends on profound reference sequences. Through linking operational taxonomic units (OTUs) with known morphospecies, we can use the deep knowledge about the autecology of these species.

Project description:For photosynthetic microbial eukaryotes, the rate-limiting step in NO3- assimilation is its reduction to nitrite (NO2-), which is catalyzed by assimilatory nitrate reductase (NR). Oceanic productivity is primarily limited by available nitrogen and, although nitrate is the most abundant form of available nitrogen in oceanic waters, little is known about the identity of microbial eukaryotes that take up nitrate. This lack of knowledge is especially severe for ice-covered seas that are being profoundly affected by climate change. To address this, we examined the distribution and diversity of NR genes in the Arctic region by way of clone libraries and data mining of available metagenomes (total of 4.24 billion reads). We directly compared NR clone phylogenies with the V4 region of the 18S rRNA gene (DNA pool) and 18S rRNA (RNA pool) at two ice-influenced stations in the Canada Basin (Beaufort Sea). The communities from the two nucleic acid templates were similar at the level of major groups, and species identified by way of NR gene phylogeny and microscopy were a subset of the 18S results. Most NR genes from arctic clone libraries matched diatoms and chromist nanoflagellates, including novel clades, while the NR genes in arctic eukaryote metagenomes were dominated by chlorophyte NR, in keeping with the ubiquitous occurrence of Mamiellophyceae in the Arctic Ocean. Overall, these data suggest that a dynamic and mixed eukaryotic community utilizes nitrate across the Arctic region, and they show the potential utility of NR as a tool to identify ongoing changes in arctic photosynthetic communities.IMPORTANCE To better understand the diversity of primary producers in the Arctic Ocean, we targeted a nitrogen cycle gene, NR, which is required for phytoplankton to assimilate nitrate into organic forms of nitrogen macromolecules. We compared this to the more detailed taxonomy from ice-influenced stations using a general taxonomic gene (18S rRNA). NR genes were ubiquitous and could be classified as belonging to diatoms, dinoflagellates, other flagellates, chlorophytes, and unknown microbial eukaryotes, suggesting novel diversity of both species and metabolism in arctic phytoplankton.

Dataset Information

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.

Publications

Accuracy of protist diversity assessments: morphology compared with cloning and direct pyrosequencing of 18S rRNA genes and ITS regions using the conspicuous tintinnid ciliates as a case study.

Similar Datasets

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