A user guide to environmental protistology: primers, metabarcoding, sequencing, and analyses

Protists - all eukaryotes besides fungi, animals, and plants - represent a major part of the taxonomic and functional diversity of eukaryotic life on the planet and drive many ecosystem processes. However, knowledge of protist communities and their diversity lags behind that of most other groups of organisms, largely due to methodological constraints. While protist communities differ markedly between habitats and biomes, they can be studied in very similar ways. Here we provide a guide to current molecular approaches used for studying protist diversity, with a particular focus on amplicon-based high-throughput sequencing (metabarcoding). We highlight that the choice of suitable primers artificially alters community profiles observed in metabarcoding studies. While there are no true universal primers to target all protist taxa as a whole, we identify some primer combinations with a wide taxonomic coverage and provide detailed information on their properties. Although environmental protistan ecological research will probably shift towards PCR-free metagenomics or/and transcriptomic approaches in a near future, metabarcoding will remain the method of choice for in-depth community analyses and taxon inventories in biodiversity surveys and ecological studies, due its great cost-efficiency, sensitivity, and throughput. In this paper we provide a guide for scientists from a broad range of disciplines to implement protists in their ecological analyses.