The morphological diversification of the species examined in this study lags behind their genetic divergence. The currently accepted 2% cutoff level of operational taxonomic unit (OTU) clustering in 18S rDNA environmental sequencing is too high to recognize genetic diversity in Asterionellopsis and very likely in many other species. Our results support the notion that a considerable number of species and diversity remain to be discovered among diatoms and that species number may be more in line with higher estimates. Molecular signatures of the species discovered here will aid in their globally consistent identification and ultimate understanding of their ecology.

DNA barcoding is a molecular tool that exploits a unique DNA sequence of a standardized gene or non-coding region for the species identification of unknown individuals. The investigation into a suitable barcode for diatoms is ongoing and there are several promising candidates including mitochondrial, plastidial and nuclear markers. We analyzed 272 sequences from 76 diatoms species in the orders Thalassiosirales, Lithodesmiales and Cymatosirales, using distance and character based approaches, to assess the applicability of a DNA barcode based on the hypervariable V4 region of the nuclear 18S rRNA gene. We show that the proposed V4 barcode separated ca. 97% of all centric diatom taxa tested using a threshold p-distance of 0.02 and that many problem pairs were further separated using a character based approach. The reliability of amplification, extensive reference library and variability seen in the V4 region make it the most promising candidate to date for a barcode marker for diatoms particularly when combined with DNA character analysis.

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