A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage‐specific barcode markers in microbial ecology

Stoeck, Thorsten; Breiner, Hans‐Werner; Filker, Sabine; Ostermaier, Veronika; Kammerlander, Barbara; Sonntag, Bettina

doi:10.1111/1462-2920.12194

Cited by 101 publications

(127 citation statements)

References 88 publications

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“…Microscopy results are shown for dimensions compared with the pore size used for sample filtering or primer mismatches is not evident, but there could be other PCR bias (for example, selective amplification of closely-related species), taxon-specific susceptibility to DNA extraction or variability in the copy number of the rDNA operon (Medinger et al, 2010;Heywood et al, 2011). This supports known inconsistencies in relative abundances estimated by HTS and microscopy for ciliates and other protists (Bachy et al, 2013;Egge et al, 2013;Santoferrara et al, 2014;Stoeck et al, 2014).…”

Section: Comparison Of Tintinnid Morphospecies and Otussupporting

confidence: 54%

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

et al. 2016

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Our knowledge on microbial biogeography depends on the way we define and study diversity. In contrast to most microbes, some protist lineages have conspicuous structures that allow comparisons of diversity concepts and measures-those based on molecules and those based on morphology. We analyzed a group of shell-bearing planktonic ciliates, the tintinnids, in a coast-to-ocean gradient using highthroughput sequencing and microscopy. First, we compared molecular operational taxonomic units (OTUs) and morphospecies in terms of assemblage composition, distribution and relationships with the environment. OTUs revealed potentially novel and rare taxa, while morphospecies showed clearer correlations with environmental factors, and both approaches coincided in supporting a coastal versus oceanic pattern. Second, we explored which processes influence assembly across the environmental gradient examined. Assemblage fluctuations were associated with significant distance-decay and changes in morphospecies size and prey proxies, thus suggesting niche partitioning as a key structuring mechanism. Our conclusion is that molecules and morphologies generally agreed, but they provided complementary data, the first revealing hidden diversity, and the latter making better connections between distribution patterns and ecological processes. This highlights the importance of linking genotypes and phenotypes (using multidisciplinary analyses and/or reliable databases of barcoded species), to understand the diversity, biogeography and ecological roles of microbes.

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Section: Comparison Of Tintinnid Morphospecies and Otussupporting

confidence: 54%

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

et al. 2016

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“…HTS surveys provide a detailed picture of the taxa present in the community, including rare species in the assemblage (18,33), and reveal diversity not evident using other methods. However, the interpretation of the HTS signal in terms of total cell abundances is not straightforward.…”

Section: Discussionmentioning

confidence: 99%

“…In addition, the few sequences obtained did not represent the dominant observed species, a clear sign of the biases in this molecular approach. More recent comparative studies used HTS, and therefore were not limited by the sequencing effort but focused on specific taxa, in particular marine and freshwater ciliates (2,(16)(17)(18). Ciliate species have the advantage of having conspicuous morphological traits…”

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confidence: 99%

Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

Giner

Forn

Romac

et al. 2016

Appl Environ Microbiol

112

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High-throughput sequencing (HTS) is revolutionizing environmental surveys of microbial diversity in the three domains of life by providing detailed information on which taxa are present in microbial assemblages. However, it is still unclear how the relative abundance of specific taxa gathered by HTS correlates with cell abundances. Here, we quantified the relative cell abundance of 6 picoeukaryotic taxa in 13 planktonic samples from 6 European coastal sites using epifluorescence microscopy on tyramide signal amplification-fluorescence in situ hybridization preparations. These relative abundance values were then compared with HTS data obtained in three separate molecular surveys: 454 sequencing of the V4 region of the 18S ribosomal DNA (rDNA) using DNA and RNA extracts (DNA-V4 and cDNA-V4) and Illumina sequencing of the V9 region (cDNA-V9). The microscopic and molecular signals were generally correlated, indicating that a relative increase in specific 18S rDNA was the result of a large proportion of cells in the given taxa. Despite these positive correlations, the slopes often deviated from 1, precluding a direct translation of sequences to cells. Our data highlighted clear differences depending on the nucleic acid template or the 18S rDNA region targeted. Thus, the molecular signal obtained using cDNA templates was always closer to relative cell abundances, while the V4 and V9 regions gave better results depending on the taxa. Our data support the quantitative use of HTS data but warn about considering it as a direct proxy of cell abundances. IMPORTANCEDirect studies on marine picoeukaryotes by epifluorescence microscopy are problematic due to the lack of morphological features and due to the limited number and poor resolution of specific phylogenetic probes used in fluorescence in situ hybridization (FISH) routines. As a consequence, there is an increasing use of molecular methods, including high-throughput sequencing (HTS), to study marine microbial diversity. HTS can provide a detailed picture of the taxa present in a community and can reveal diversity not evident using other methods, but it is still unclear what the meaning of the sequence abundance in a given taxon is. Our aim is to investigate the correspondence between the relative HTS signal and relative cell abundances in selected picoeukaryotic taxa. Environmental sequencing provides reasonable estimates of the relative abundance of specific taxa. Better results are obtained when using RNA extracts as the templates, while the region of 18S ribosomal DNA had different influences depending on the taxa assayed. Protists are key components of marine ecosystems, being major players in the global respiration and production budgets (1, 2) and playing central roles in marine food webs (3). Despite their importance and ubiquity, it was only during the past decade that environmental studies, based on molecular (i.e., culture-independent) techniques, revealed an unsuspected protist diversity in a large variety of marine ecosystems (4-13). These studies we...

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“…Stoeck et al (2014) found many quantitative and qualitative differences between amplicons and morphotypes. They saw that taxon abundance from amplicon data did not necessarily track morphotype abundance.…”

Section: Discussionmentioning

confidence: 99%

Ciliate micrograzer dynamics of the New England Shelf

Brownlee¹

2017

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Protists play important roles in grazing and nutrient recycling, but quantifying these roles has been hindered by difficulties in collecting, culturing, and observing these often-delicate cells. During long-term deployments at the Martha's Vineyard Coastal Observatory (MVCO) (Massachusetts, USA), Imaging FlowCytobot (IFCB) made it possible to study live cells in situ without the need to culture or preserve. IFCB records images of cells with chlorophyll fluorescence above a trigger threshold, so taxonomically resolved analysis of protists is limited to mixotrophs and herbivores, which have eaten recently. To overcome this limitation, I coupled a broad-application 'live cell' fluorescent stain with a modified IFCB so that protists which do not contain chlorophyll (such as consumers of unpigmented bacteria and other heterotrophs) can also be recorded. Staining IFCB (IFCB-S) revealed higher abundances of grazers than the original IFCB, as well as some cell types not previously detected. To analyze a 10-year time series of herbivorous ciliates at MVCO and address broad patterns of seasonality of major ciliate classes and their components, I employed a statistical model that estimates a seasonal density pattern and simultaneously accounts for and separates any annual-scale effects. I describe the seasonality of three functional groups: a phototrophic ciliate, a mixotroph, and a group of strict heterotrophs, and comment on potential drivers of these patterns. DNA sequencing has also contributed to the study of protist communities, providing new insight into diversity, predator-prey interactions, and discrepancies between morphologically defined species and genotype. To explore how well IFCB images can be used to detect seasonal community change of the class Spirotrichea, an important and numerous group, I used high-throughput sequencing (HTS), which does not discriminate between chlorophyll-containing cells and the rest of the community. I report on species and genera of ciliates for which morphotype and genotype displayed high congruency. In comparing how well temporal aspects of genotypes and morphotypes correspond, I found that HTS was critical to detect and identify certain ciliates occupying a niche associated with warmer temperatures. I further showed that when these types of analyses are combined with IFCB results, they can provide hypotheses about food preferences. These last six and a half years have been full of scientific and self-discovery. I could not have pursued this adventure without a village of support. I'd like to thank all of these people.To my advisor, Heidi, your guidance, patience, and belief in me is something I will always cherish and never forget. Apart from being an amazing advisor in a academic way (opening my eyes to diverse questions, guiding my writing, etc.), you also helped me with many life obstacles and stimulated me during my difficult moments. I am forever grateful I had you as role model throughout these years and for that I am a better scientist.To Rob, thank you for takin...

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A morphogenetic survey on ciliate plankton from a mountain lake pinpoints the necessity of lineage‐specific barcode markers in microbial ecology

Cited by 101 publications

References 88 publications

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

Patterns and processes in microbial biogeography: do molecules and morphologies give the same answers?

Environmental Sequencing Provides Reasonable Estimates of the Relative Abundance of Specific Picoeukaryotes

Ciliate micrograzer dynamics of the New England Shelf

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