Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐r<scp>DNA</scp> in Environmental Microbial Eukaryotic Diversity Surveys

Mahé, Frédéric; Mayor, Jordan R.; Bunge, John; Chi, Jingyun; Siemensmeyer, Tobias; Stoeck, Thorsten; Wahl, Benjamin; Paprotka, Tobias; Filker, Sabine; Dunthorn, Micah

doi:10.1111/jeu.12187

Cited by 56 publications

(37 citation statements)

References 49 publications

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“…The combined samples were amplified with a high fidelity polymerase for the V4 region of 18S rRNA following ref. 52 with universal eukaryotic V4 primers (TAReuk454FWD1 and TAReukREV3) 13 . This primer pair has not shown a preference for amplifying Apicomplexa, and the V4 primers were able to amplify all Oomycota in GenBank in an in silico analysis.…”

Section: Discussionmentioning

confidence: 99%

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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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Section: Discussionmentioning

confidence: 99%

“…Following ref. 52 , assembled paired-end reads were filtered using Cutadapt 58 v1.9 and retained if they had both primers and no ambiguously named nucleotides (Ns). Reads were dereplicated into strictly identical amplicons with VSEARCH 59 v1.6.0, then clustered using Swarm 16 v2.1.5, with the parameter d = 1 and the fastidious option on.…”

Section: Marine Samplesmentioning

confidence: 99%

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

et al. 2017

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“…f Kraków, Opatkowice (pond), 2 sampling points. g Kraków, Rajsko (pond), 2 sampling points examination of protistan diversity (Mahe et al 2015) through the detection of species or molecular operational taxonomic units (MOTU) composition. However, for the obtained data to be useful for identifying new species, they must be matched with previously described taxons by comparing them with sequences deposited in databases like GenBank or BOLD (Thomsen and Willerslev 2015).…”

Section: Paramecium Biaurelia As a Good Model For Testing Microbial Bmentioning

confidence: 99%

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate Paramecium biaurelia (P. aurelia species complex, Ciliophora, Protozoa)

2018

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Species (or cryptic species) identification in microbial eukaryotes often requires a combined morphological and molecular approach, and if possible, mating reaction tests that confirm, for example, that distant populations are in fact one species. We used P. biaurelia (one of the 15 cryptic species of the P. aurelia complex) collected worldwide from 92 sampling points over 62 years and analyzed with the three above mentioned approaches as a model for testing protistan biogeography hypotheses. Our results indicated that despite the large distance between them, most of the studied populations of P. biaurelia do not differ from each other (rDNA fragment), or differ only slightly (COI mtDNA fragment). These results could suggest that in the past, the predecessors of the present P. biaurelia population experienced a bottleneck event, and that its current distribution is the result of recent dispersal by natural or anthropogenic factors. Another possible explanation for the low level of genetic diversity despite the huge distances between the collecting sites could be a slow rate of mutation of the studied DNA fragments, as has been found in some other species of the P. aurelia complex. COI haplotypes determined from samples obtained during field research conducted in 2015-2016 in 28 locations/374 sampling points in southern Poland were shared with other, often distant P. biaurelia populations. In the Kraków area, we found 5 of the 11 currently known COI P. biaurelia haplotypes. In 5 of 7 reservoirs from which P. biaurelia was obtained, two different COI haplotypes were identified.

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“…Users can now dereplicate their sequencing reads into strictly identical amplicons (sensu Mahé et al (2015); i.e., reads that have exactly the same sequences with no mutations, insertions, deletions). This fast dereplication approach uses the same algorithm as in VSEARCH (https://github.com/torognes/vsearch).…”

Section: Other New and Useful Featuresmentioning

confidence: 99%

Swarm v2: highly-scalable and high-resolution amplicon clustering

Mahé

Rognes

Quince

et al. 2015

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Previously we presented Swarm v1, a novel and open source amplicon clustering program that produced fine-scale molecular operational taxonomic units (OTUs), free of arbitrary global clustering thresholds and input-order dependency. Swarm v1 worked with an initial phase that used iterative single-linkage with a local clustering threshold (d), followed by a phase that used the internal abundance structures of clusters to break chained OTUs. Here we present Swarm v2 that has two important novel features: 1) a new algorithm for d = 1 that allows the computation time of the program to scale linearly with increasing amounts of data; and 2) the new fastidious option that reduces under-grouping by grafting low abundant OTUs (e.g., singletons and doubletons) onto larger ones. Swarm v2 also directly integrates the clustering and breaking phases, dereplicates sequencing reads with d = 0, outputs OTU representatives in fasta format, and plots individual OTUs as two-dimensional networks.

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Comparing High‐throughput Platforms for Sequencing the V4 Region of SSU‐rDNA in Environmental Microbial Eukaryotic Diversity Surveys

Cited by 56 publications

References 49 publications

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

Parasites dominate hyperdiverse soil protist communities in Neotropical rainforests

Worldwide sampling reveals low genetic variability in populations of the freshwater ciliate Paramecium biaurelia (P. aurelia species complex, Ciliophora, Protozoa)

Swarm v2: highly-scalable and high-resolution amplicon clustering

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