title: DNA metabarcoding reveals that 200-μm-size-fractionated filtering is unable to discriminate between planktonic microbial and large eukaryotes.
abstract: Microeukaryotic plankton (0.2-200 μm) are critical components of aquatic ecosystems and key players in global ecological processes. High-throughput sequencing is currently revolutionizing their study on an unprecedented scale. However, it is currently unclear whether we can accurately, effectively and quantitatively depict the microeukaryotic plankton communities using traditional size-fractionated filtering combined with molecular methods. To address this, we analysed the eukaryotic plankton communities both with, and without, prefiltering with a 200 μm pore-size sieve -by using SSU rDNA-based high-throughput sequencing on 16 samples with three replicates in each sample from two subtropical reservoirs sampled from January to October in 2013. We found that ~25% reads were classified as metazoan in both size groups. The species richness, alpha and beta diversity of plankton community and relative abundance of reads in 99.2% eukaryotic OTUs showed no significant changes after prefiltering with a 200 μm pore-size sieve. We further found that both >0.2 μm and 0.2-200 μm eukaryotic plankton communities, especially the abundant plankton subcommunities, exhibited very similar, and synchronous, spatiotemporal patterns and processes associated with almost identical environmental drivers. The lack of an effect on community structure from prefiltering suggests that environmental DNA from larger metazoa is introduced into the smaller size class. Therefore, size-fractionated filtering with 200 μm is insufficient to discriminate between the eukaryotic plankton size groups in metabarcoding approaches. Our results also highlight the importance of sequencing depth, and strict quality filtering of reads, when designing studies to characterize microeukaryotic plankton communities.
authors.affiliation: Aquatic EcoHealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.
authors.forename: Lemian
authors.initials: L
authors.lastname: Liu
authors.affiliation: Aquatic EcoHealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.|University of Chinese Academy of Sciences, Beijing, 100049, China.
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.affiliation: School of Natural Science and Psychology, Liverpool John Moores University, Byrom Street, Liverpool, L3 3AF, UK.|School of Life Sciences, University of Lincoln, Lincoln, LN6 7TS, UK.
authors.forename: David M
authors.initials: DM
authors.lastname: Wilkinson
authors.affiliation: Aquatic EcoHealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.
authors.forename: Huihuang
authors.initials: H
authors.lastname: Chen
authors.affiliation: Aquatic EcoHealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.
authors.forename: Xiaoqing
authors.initials: X
authors.lastname: Yu
authors.affiliation: Aquatic EcoHealth Group, Key Laboratory of Urban Environment and Health, Institute of Urban Environment, Chinese Academy of Sciences, Xiamen, 361021, China.
authors.forename: Jun
authors.identifier: http://orcid.org/0000-0002-7920-2777
authors.initials: J
authors.lastname: Yang
mesh_terms: D016000:Cluster Analysis; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D056890:Eukaryota / Q000145:classification* / Q000235:genetics*; D005374:Filtration / Q000379:methods*; D056186:Metagenomics / Q000379:methods*; D010802:Phylogeny; D010933:Plankton / Q000145:classification* / Q000235:genetics*; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D017422:Sequence Analysis, DNA
chemical_list: D004275:DNA, Ribosomal; D012337:RNA, Ribosomal, 18S
keywords: 
  eDNA
  ; eukaryotic planktonic community; high-throughput sequencing; size-fractionated filtering; subtropical reservoir