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

Abstract

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.

Alveolates (dinoflagellates, ciliates and apicomplexans) and Rhizarians are the most common microbial eukaryotes in temperate Appalachian karst caves

“…This lab has been collecting plankton from surface water samples from various sites in and around Southwestern Virginia (the location of Panel and Bolling Caves) since 2015 and has an archive of freshwater 18S rDNA sequences which, to date, has 3283 unique ASVs (Cahoon et al ., 2018, Fawley et al ., 2021, and unpublished data). Only 2.5% of the cave samples matched one of these sequences with greater than 99% identity.…”

Metabarcoding reveals unique microbial mat communities and evidence of biogeographic influence in low‐oxygen, high‐sulfur sinkholes and springs

“…The rbc L marker revealed a diverse array of diatom taxa with high taxonomic resolution. This study adds to previous metabarcoding research that has used the rbc L marker to successfully characterize algal communities (e.g., Fawley et al., 2021 ; Pérez‐Burillo et al., 2022 ; Wolf & Vis, 2019 ). Culturing diatoms from our samples proved to be an important and successful method to improve the accuracy of taxonomic assignment.…”

Unrecognized diversity and distribution of soil algae from Maritime Antarctica (Fildes Peninsula, King George Island)

“…Those amplicons revealed an astonishing diversity of OTUs of both classes. A higher diversity at the species level may be recovered when combining PCR amplicon metabarcoding with specific lineage-targeted primers (e.g., Fawley et al, 2021 ). The specific primer combination for Xanthophyceae was particularly successful as almost no other sequences (e.g., fungi or other eukaryotes) have been amplified.…”