Finding needles in a haystack-Extensive diversity in the eustigmatophyceae revealed by community metabarcode analysis targeting the rbcL gene using lineage-directed primers.

Sequences from the Stramenopile class Eustigmatophyceae are rarely reported in metabarcoding studies, and when they have been reported, there are very few haplotypes. We hypothesized that the paucity of eustigmatophyte species detected in these studies may be a result of the metabarcoding techniques used, which have primarily employed universal ribosomal RNA gene regions. In this study, we examined environmental DNA samples from 22 sites in southwestern Virginia, some of which had previously been studied using ribosomal RNA analysis. We used metabarcoding techniques targeting the plastid rbcL gene with new primers designed to produce a 370 bp amplicon from all lineages of the Eustigmatophyceae in a reference collection. The amplicons were then analyzed with DADA2 to produce amplicon sequence variants (ASVs). Our results revealed 184 rbcL haplotypes that can be tentatively assigned to the Eustigmatophyceae from these sites, representing much higher diversity than has been detected by ribosomal DNA-based studies. The techniques employed can be used for future studies of population structure, ecology, distribution, and diversity of this class. With these techniques, it should be possible to make realistic estimates of the species-level diversity of the Eustigmatophyceae on local, regional, and perhaps global scales.