Abstract
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.
Highlights
Diatoms are ubiquitous and morphological species are widely used to identify environmental changes in both freshwater and marine ecosystems [1]
Some strains, including all of the Cymatosirales and Skeletonema marinoi, and S. menzelli needed to be amplified a second time from the first PCR product to ensure sufficient DNA for sequencing
These two species amplified and sequenced well for the V4 region, there were two bands visualized in the gels used to verify the amplification steps with one of the bands 450 bp, which was the target length and the second band approximately 600 bp
Summary
Diatoms are ubiquitous and morphological species are widely used to identify environmental changes in both freshwater and marine ecosystems [1]. Especially the thalassiosiroids are responsible for much of the primary production throughout the world oceans [2]. A rapid means of correct species identification is essential to fully exploit high throughput environmental sequence surveys, archived material, including ancient DNA, and data from clone libraries of uncultivated eukaryotes. Accurate identification of diatoms will facilitate expanded monitoring efforts in the face of natural versus anthropogenic changes in marine and freshwater ecosystems. The high number of described morpho-species and an ever increasing number of newly discovered semi-cryptic and cryptic species adds to the challenges of expediency in the routine identification of diatoms. In the last decade or so, nearly every study applying a combination of molecular and classical methods to taxonomically re-apprise ‘‘difficult’’ species resulted in discovery of species complexes [3], [4], [5], highlighting the need to revise interpretations of previous findings in microbial population and community ecology and geography
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