Benthic protists: the under-charted majority

Forster, Dominik; Dunthorn, Micah; Mahé, Frédéric; Dolan, John R.; Audic, Stéphane; Bass, David; Bittner, Lucie; Boutte, Christophe; Christen, Richard; Claverie, Jean‐Michel; Decelle, Johan; Edvardsen, Bente; Egge, Elianne; Eikrem, Wenche; Gobet, Angélique; Kooistra, Wiebe H. C. F.; Logares, Ramiro; Massana, Ramón; Montresor, Marina; Not, Fabrice; Ogata, Hiroyuki; Pawłowski, Jan; Pernice, Massimo C.; Romac, Sarah; Shalchian-Tabrizi, Kamran; Simon, Nathalie; Richards, Thomas A.; Santini, Sébastien; Sarno, Diana; Siano, Raffaele; Vaulot, Daniel; Wincker, Patrick; Zingone, Adriana; Vargas, Colomban de; Stoeck, Thorsten

doi:10.1093/femsec/fiw120

Cited by 88 publications

(75 citation statements)

References 74 publications

(102 reference statements)

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“…This has already been demonstrated in assessing microbial eukaryote communities, using the small subunit ribosomal RNA gene (SSU rRNA) as a marker (reviewed in [11], but also see [9,13,18,19]). As yet, we have only skimmed the surface of microbial benthic diversity [20], many basic aspects of experimental design remain poorly known, and the methodologic pipelines are not sufficiently standardised.…”

Section: Introductionmentioning

confidence: 99%

DNA extraction replicates improve diversity and compositional dissimilarity in metabarcoding of eukaryotes in marine sediments

Lanzén¹,

Lekang²,

Jonassen³

et al. 2017

PLoS ONE

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Human impact on marine benthic communities has traditionally been assessed using visible morphological traits and has focused on the macrobenthos, whereas the ecologically important organisms of the meio- and microbenthos have received less attention. DNA metabarcoding offers an alternative to this approach and enables a larger fraction of the biodiversity in marine sediments to be monitored in a cost-efficient manner. Although this methodology remains poorly standardised and challenged by biases inherent to rRNA copy number variation, DNA extraction, PCR, and limitations related to taxonomic identification, it has been shown to be semi-quantitative and useful for comparing taxon abundances between samples. Here, we evaluate the effect of replicating genomic DNA extraction in order to counteract small scale spatial heterogeneity and improve diversity and community structure estimates in metabarcoding-based monitoring. For this purpose, we used ten technical replicates from three different marine sediment samples. The effect of sequence depth was also assessed, and in silico pooling of DNA extraction replicates carried out in order to maintain the number of reads constant. Our analyses demonstrated that both sequencing depth and DNA extraction replicates could improve diversity estimates as well as the ability to separate samples with different characteristics. We could not identify a “sufficient” replicate number or sequence depth, where further improvements had a less significant effect. Based on these results, we consider replication an attractive alternative to directly increasing the amount of sample used for DNA extraction and strongly recommend it for future metabarcoding studies and routine assessments of sediment biodiversity.

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Section: Introductionmentioning

confidence: 99%

DNA extraction replicates improve diversity and compositional dissimilarity in metabarcoding of eukaryotes in marine sediments

Lanzén¹,

Lekang²,

Jonassen³

et al. 2017

PLoS ONE

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“…For example, while the broadly targeted V4 primers used here amplify a wide variety of eukaryotic lineages 13,[25][26][27][28][29] , other primers could have amplified additional taxa [30][31][32] (Supplementary Table 1). The resolution power of metabarcoding also has its limits.…”

Section: Discussionmentioning

confidence: 99%

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

et al. 2017

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High animal and plant richness in tropical rainforest communities has long intrigued naturalists. It is unknown if similar hyperdiversity patterns are reflected at the microbial scale with unicellular eukaryotes (protists). Here we show, using environmental metabarcoding of soil samples and a phylogeny-aware cleaning step, that protist communities in Neotropical rainforests are hyperdiverse and dominated by the parasitic Apicomplexa, which infect arthropods and other animals. These host-specific parasites potentially contribute to the high animal diversity in the forests by reducing population growth in a density-dependent manner. By contrast, too few operational taxonomic units (OTUs) of Oomycota were found to broadly drive high tropical tree diversity in a host-specific manner under the Janzen-Connell model. Extremely high OTU diversity and high heterogeneity between samples within the same forests suggest that protists, not arthropods, are the most diverse eukaryotes in tropical rainforests. Our data show that protists play a large role in tropical terrestrial ecosystems long viewed as being dominated by macroorganisms.S ince the works of early naturalists such as von Humboldt and Bonpland 1 , we have known that animal and plant communities in tropical rainforests are exceedingly species rich. For example, one hectare can contain more than 400 tree species 2 and one tree can harbour more than 40 ant species 3 . This hyperdiversity of trees has been partially explained by the Janzen-Connell model 4,5 , which hypothesizes that host-specific predators and parasites reduce plant population growth in a density-dependent manner 6,7 . Sampling up in the tree canopies and below on the ground has further led to the view that arthropods are the most diverse eukaryotes in tropical rainforests 8,9 .The focus on eukaryotic macroorganisms in these studies is primarily because they are familiar and readily observable to us. We do not know whether the less familiar and less readily observable protists-microbial eukaryotes that are not animals, plants or fungi 10 -inhabiting these same ecosystems exhibit similar diversity patterns. To evaluate if macroorganismic diversity patterns are reflected at the microbial scale with protists, we conducted an environmental DNA metabarcoding study by sampling soils in 279 locations in a variety of lowland Neotropical forest types in La Selva Biological Station, Costa Rica, Barro Colorado Island, Panama and Tiputini Biodiversity Station, Ecuador. This metabarcoding approach has the power to uncover known and new taxa on a massive scale 11 . By amplifying DNA extracted from the soils with broadly targeted primers for the V4 region of 18S rRNA and sequencing it using the Illumina MiSeq platform, we were able to detect most eukaryotic lineages, and assess the diversity and relative dominance of free-living and parasitic lineages.

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“…In practice, this only works to a limited extent as is shown in the observed high species numbers in high‐throughput metabarcoding studies (e.g., Filker et al ) and also in this study. A commonly applied sequence divergence for ciliate V4 fragments to differentiate between species is 3%, but sometimes also 1%, 2%, and 5% are used (Caron et al ; Nebel et al ; Dunthorn et al ; Weber and Pawlowski ; Balzano et al ; Massana et al ; Forster et al ). Our study suggested that 3% is indeed a proper cutoff for the V4 fragment of most ciliate species, whose intragenomic variability was generally < 3%, while their interspecies variability was higher.…”

Section: Discussionmentioning

confidence: 99%

“…As protist taxa evolve at different rates, there is no consensus on the similarity threshold value. Most commonly, values between 95% and 97% are applied to discriminate between protist taxa (Kammerlander et al ; Massana et al ; Forster et al ; Zhao and Xu , ). USEARCH, in a first step, selects one amplicon from an amplicon pool to serve as a centroid, and then the remaining amplicons are pairwise compared to the centroid (Edgar , ).…”

Section: Materials and Proceduresmentioning

confidence: 99%

“…The advent of high‐throughput DNA sequencing has revolutionized the study on the diversity of microbial eukaryotes. The application of deep‐sequencing techniques in combination with genetic barcode markers, such as the widely preferred hypervariable V4 and V9 fragments of the 18S rRNA gene, revealed extensive diversity of microeukaryotes in various marine environments (de Vargas et al ; Kammerlander et al ; Massana et al ; Forster et al ; Filker et al ; Mahé et al ). The extremely high diversity of the 18S rRNA gene largely exceeded species diversity detected by morphological methods.…”

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confidence: 99%

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Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single‐cell high‐throughput DNA sequencing

Zhao

Filker

et al. 2019

Limnology & Ocean Methods

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Diversity assessments using the small subunit (SSU) rRNA gene allow for rapid assessments of microeukaryotic communities. Yet, for microeukaryotes, only limited information is available on the extent to which sequence variations within individual genomes exist at this locus. We investigated 14 marine species belonging to four major ciliate groups by single‐cell high‐throughput DNA sequencing to estimate the extent of intragenomic polymorphism in the V4 region of the SSU rRNA gene. Based on these results, we evaluated the potential effect of polymorphism on metabarcoding studies. The intragenomic variability was generally below 3% among about 1000 unique sequences obtained from each studied cell, except of Protocruzia sp., whose intragenomic variability was as high as 15.6% among about 2000 unique sequences. Intragenomic variations significantly correlated with the number of macronuclei, but not with the biovolume of cell or macronuclei, indicating the occurrence of nonuniform evolution within the macronuclei. The removal of rare unique sequences greatly decreased the number of variations. For diversity studies, we therefore propose to conservatively discard rare amplicons. Considering the intragenomic variation within the V4 region, a sequence similarity cutoff of 3% to delimit marine ciliate operational taxonomic units could be substantiated, as at least this threshold could exclude most effect of intragenomic polymorphisms, although it may be too strict for some particular groups.

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Benthic protists: the under-charted majority

Cited by 88 publications

References 74 publications

DNA extraction replicates improve diversity and compositional dissimilarity in metabarcoding of eukaryotes in marine sediments

DNA extraction replicates improve diversity and compositional dissimilarity in metabarcoding of eukaryotes in marine sediments

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

Effects of intragenomic polymorphism in the SSU rRNA gene on estimating marine microeukaryotic diversity: A test for ciliates using single‐cell high‐throughput DNA sequencing

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