Not all are free‐living: high‐throughput DNA metabarcoding reveals a diverse community of protists parasitizing soil metazoa

Abstract

Protists, the most diverse eukaryotes, are largely considered to be free-living bacterivores, but vast numbers of taxa are known to parasitize plants or animals. High-throughput sequencing (HTS) approaches now commonly replace cultivation-based approaches in studying soil protists, but insights into common biases associated with this method are limited to aquatic taxa and samples. We created a mock community of common free-living soil protists (amoebae, flagellates, ciliates), extracted DNA and amplified it in the presence of metazoan DNA using 454 HTS. We aimed at evaluating whether HTS quantitatively reveals true relative abundances of soil protists and at investigating whether the expected protist community structure is altered by the co-amplification of metazoan-associated protist taxa. Indeed, HTS revealed fundamentally different protist communities from those expected. Ciliate sequences were highly over-represented, while those of most amoebae and flagellates were under-represented or totally absent. These results underpin the biases introduced by HTS that prevent reliable quantitative estimations of free-living protist communities. Furthermore, we detected a wide range of nonadded protist taxa probably introduced along with metazoan DNA, which altered the protist community structure. Among those, 20 taxa most closely resembled parasitic, often pathogenic taxa. Therewith, we provide the first HTS data in support of classical observational studies that showed that potential protist parasites are hosted by soil metazoa. Taken together, profound differences in amplification success between protist taxa and an inevitable co-extraction of protist taxa parasitizing soil metazoa obscure the true diversity of free-living soil protist communities.

Random sampling causes the low reproducibility of rare eukaryotic OTUs in Illumina COI metabarcoding

“…A total of 20 sessile and 13 protist taxa were likely present on the legs of spider crabs and other arthropods. In fact, epiphytes, endosymbionts and also parasites are commonly described in sequence libraries (Geisen et al, 2015; de Vargas et al, 2015). Motile OTUs (e.g., among arthropods, molluscs and echinoderms) may be part of the diet of carnivorous annelids and thus present in the section of their digestive tract that was used for DNA extraction.…”

Detection of rare and invasive freshwater fish species using eDNA pyrosequencing: Lake Iznik ichthyofauna revised

“…This method is known as eDNA metabarcoding . eDNA metabarcoding has been used in many studies including detection of fish species (Thomsen et al, 2012a,b;Kelly et al, 2014;Evans et al, 2016), aquatic plants (Cowart et al, 2015;Visco et al, 2015), macroinvertebrates (Elbrecht and Leese, 2015), amphibians (Evans et al, 2016), arthropods (Yu et al, 2012), protists (Pawlowski et al, 2014;Geisen et al, 2015;Kosakyan et al, 2015), soil biodiversity (Bienert et al, 2012;Taberlet et al, 2012;Yoccoz et al, 2012), diet assessment (Valentini et al, 2009;Deagle et al, 2013;De Barba et al, 2014) and ballast water surveillance (Zaiko et al, 2015). This study presents the first eDNA metabarcoding results of an entire lake ichthyofauna.…”

Can Theory Improve the Scope of Quantitative Metazoan Metabarcoding?

“…Though there are concerns about how well read/sequence frequency reflects the relative abundance of populations in an environmental sample-driven largely by differential amplification success of target genomes [3,[7][8][9]12,13], it is worth noting that at least two recent studies-one on nematodes and one on marine fouling communities-found a strong correlation between the proportion of metabarcoding reads from a taxon and the relative abundance of that taxon given point counts [31,39], as well as a strong correlation with taxon frequency from individual barcode data. If this relationship is supported in further studies, and if we can use information such as the number of haplotypes in a taxon as complementary information for minimum abundance, then we may start to improve on our ability to recover actual ecology from actual molecules.…”

“…Next-generation sequencing (NGS) has permitted the now-commonplace exploration of fungal, bacterial, and viral diversity by generating 10 5 -10 7 sequence reads per sample and using barcoding approaches (match of sequence to known taxonomic samples for that genomic region) to identify the lineages, OTUs, or species present and their relative abundance. While there is no doubt that this has transformed our understanding of functional ecosystem processes and ecology of microbes, other prokaryotes, and microscopic eukaryotes at this scale [3][4][5][6][7], there are definite limitations-including the difficulty of reliably matching molecular sequence data to described species or taxonomic diversity [8,9]. For example, some taxa (e.g.…”