Improved 18S rDNA amplification protocol for assessing protist diversity in oxygen-deficient marine systems

High-throughput sequencing techniques have been increasingly used in biodiversity estimates and in the detection of rare species within prokaryotic and eukaryotic assemblages in different habitats. In studies of protists, the V4 region of the 18S rRNA gene is the genetic marker most often used. However, established primers for the variable region V4 show several mismatches when used to examine protist groups important in oxygen-deficient aquatic systems, such as euglenozoans, some stramenopiles, and ciliates (e.g. mesodiniids). In this study, we designed new general primers covering the V4 and V5 region and used them in combination with a primer mix in a 2-step PCR approach to improve our understanding of eukaryotic diversity in oxygen-deficient zones. Prior to amplification of environmental samples from oxygen-deficient water layers of the Black Sea, we tested and improved the protocol on a protist mock community (1 ciliate and 5 flagellate species). With the developed approach, we detected protist taxa of a broad taxonomic range such as several ciliate groups including the hitherto missing mesodiniids, as well as of dinoflagellates, apicomplexans, apusomonads, different stramenopiles (bicosoecids, diatoms, chrysophytes, several marine stramenopile clades, bolidophytes), and haptophytes, excavates (jakobids, different otherwise missing euglenozoan groups, and schizopyrenids), rhizarians, amoebozoans, and choanoflagellates in the Black Sea sample. Therefore, we conclude that the new primers and tested amplification protocol improve the assessment of protist diversity from oxygen-deficient waters including phylogenetic groups that could not be detected by most published primers.