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Research article - Peer-reviewed, 2022

Effects of operational taxonomic unit inference methods on soil microeukaryote community analysis using long-read metabarcoding

Sahraei, Shadi Eshghi; Furneaux, Brendan; Kluting, Kerri; Zakieh, Mustafa; Rydin, Hakan; Hytteborn, Hakan; Rosling, Anna


Long amplicon metabarcoding has opened the door for phylogenetic analysis of the largely unknown communities of microeukaryotes in soil. Here, we amplified and sequenced the ITS and LSU regions of the rDNA operon (around 1500 bp) from grassland soils using PacBio SMRT sequencing. We tested how three different methods for generation of operational taxonomic units (OTUs) effected estimated richness and identified taxa, and how well large-scale ecological patterns associated with shifting environmental conditions were recovered in data from the three methods. The field site at Kungsangen Nature Reserve has drawn frequent visitors since Linnaeus's time, and its species rich vegetation includes the largest population of Fritillaria meleagris in Sweden. To test the effect of different OTU generation methods, we sampled soils across an abrupt moisture transition that divides the meadow community into a Carex acuta dominated plant community with low species richness in the wetter part, which is visually distinct from the mesic-dry part that has a species rich grass-dominated plant community including a high frequency of F. meleagris. We used the moisture and plant community transition as a framework to investigate how detected belowground microeukaryotic community composition was influenced by OTU generation methods. Soil communities in both moisture regimes were dominated by protists, a large fraction of which were taxonomically assigned to Ciliophora (Alveolata) while 30%-40% of all reads were assigned to kingdom Fungi. Ecological patterns were consistently recovered irrespective of OTU generation method used. However, different methods strongly affect richness estimates and the taxonomic and phylogenetic resolution of the characterized community with implications for how well members of the microeukaryotic communities can be recognized in the data.


clustering; ITS; rDNA; species hypothesis

Published in

Ecology and Evolution
2022, volume: 12, number: 3, article number: e8676
Publisher: WILEY

Authors' information

Sahraei, Shadi Eshghi
Uppsala University
Furneaux, Brendan
Uppsala University
Kluting, Kerri
Uppsala University
Zakieh, Mustafa
Uppsala University
Swedish University of Agricultural Sciences, Department of Plant Breeding
Rydin, Hakan
Uppsala University
Hytteborn, Hakan
Uppsala University
Rosling, Anna
Uppsala University

UKÄ Subject classification

Biological Systematics

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