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.

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Non-indigenous species (NIS) in the Arctic have an increased likelihood of arrival from ship traffic in the region, while the survival potential of the species becomes more likely in a warming environment. Monitoring is essential to detect the rate and magnitude of the establishment of NIS. In this study, a list of 123 potential marine NIS for Svalbard was drafted and the presence of marine NIS in soft sediment of Kongsfjorden in Svalbard was assessed using molecular metabarcoding techniques. For 37 species, including eight potential Arctic NIS, we generated new 18S and/or COI barcode sequences to improve the available online reference databases. In total, 299 species were identified in the sediment samples, including seven potential NIS. Three of these potential NIS have not been reported before in Svalbard: the harpacticoid copepod Euterpina acutifrons, and the ascidians Botrylloides violaceus and Molgula manhattensis. Another novel observation for Svalbard was the polychaete Chone mollis. Additional studies are needed to assess whether the NIS have been established on Svalbard and what their potential impact on the local system may be. Metabarcoding proved to be an effective monitoring tool to detect the presence of new species in Svalbard marine waters. We advise its use to set up a baseline record for the presence of NIS at points of entry, especially harbours. This approach is also valuable for biodiversity monitoring, in particular the detection of small organisms and life stages that are hard to identify using current visual techniques.