A comparison of molecular markers and morphology for Neidium taxa (Bacillariophyta) from eastern North America.

Historically, a morphological species concept has applied shape subjectively in the delimitation of diatom species. This has led to confusion between taxa within the benthic diatom genus Neidium. Samples from Ontario, Quebec, Nova Scotia, Newfoundland (Canada) and New York (USA) were examined for Neidium taxa under LM and SEM. Fourier shape analysis showed that shape as a taxonomic character was not able to discern all species. Isolated individuals from the samples were amplified and sequenced for three chloroplast molecular markers (rbcL, psbC, and psbA) and one nuclear ribosomal molecular marker (18S). Phylogenetic reconstructions were completed with the concatenated chloroplast and 18S dataset using Maximum Likelihood and Bayesian analyses. The concatenated chloroplast dataset exhibited a species-level resolution phylogeny of Neidium taxa. The 18S dataset had a lower level of sequence divergence and was unable to differentiate between Neidium taxa. We present emended species descriptions and sequence data for four previously described species: Neidium sacoense, N. longiceps, N. fossum, and N. affine. We describe three novel species (Neidium lowei, N. promontorium, and N. potapovae) and identify two forms with unique molecular signatures. The distinguishing features of N. lowei are its size, valve shape, and longitudinal canal structure. Distinguishing features of N. promontorium are its valve shape, longitudinal canal and apex formation, and surface depression along the axial area. Neidium potapovae is distinguished by its size, formation of valve and apices and single longitudinal canal. This paper demonstrates how future phylogenetic treatments using single cell multigene sequencing can help resolve taxonomic confusion within diatoms.

Single cell PCR amplification of diatoms using fresh and preserved samples.

Single cell Chelex® DNA extraction and nested PCR amplification were used to examine partial gene sequences from natural diatom populations for taxonomic and phylogenetic studies at and above the level of species. DNA was extracted from cells that were either fresh collected or stored in RNAlater. Extractions from Lugol's fixation were also attempted with limited success. Three partial gene sequences (rbcL, 18S, and psbA) were recovered using existing and new primers with a nested or double nested PCR approach with amplification and success rates between 70 and 96%. An rbcL consensus tree grouped morphologically similar specimens and was consistent across the two primary sample treatments: fresh and RNAlater. This tool will greatly enhance the number of microscopic diatom taxa (and potentially other microbes) available for barcoding and phylogenetic studies. The near-term increase in sequence data for diatoms generated via routine single cell extractions and PCR will act as a multiproxy validation of longer-term next generation genomics.