High-Level Diversity of Dinoflagellates in the Natural Environment, Revealed by Assessment of Mitochondrial cox1 and cob Genes for Dinoflagellate DNA Barcoding

Abstract

DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA. An effective DNA barcoding marker would be very helpful for unraveling the poorly understood species diversity of dinoflagellates in the natural environment. In this study, the potential utility for DNA barcoding of mitochondrial cytochrome c oxidase 1 (cox1) and cytochrome b (cob) was assessed. Among several primer sets examined, the one amplifying a 385-bp cob fragment was most effective for dinoflagellates. This short cob fragment is easy to sequence and yet possess reasonable taxon resolution. While the lack of a uniform gap between interspecific and intraspecific distances poses difficulties in establishing a phylum-wide speciesdiscriminating distance threshold, the variability of cob allows recognition of species within particular lineages. The potential of this cob fragment as a dinoflagellate species marker was further tested by applying it to an analysis of the dinoflagellate assemblages in Long Island Sound (LIS) and Mirror Lake in Connecticut. In LIS, a highly diverse assemblage of dinoflagellates was detected. Some taxa can be identified to the species and some to the genus level, including a taxon distinctly related to the bipolar species Polarella glacialis, and the large number of others cannot be clearly identified, due to the inadequate database. In Mirror Lake, a Ceratium species and an unresolved taxon were detected, exhibiting a temporal transition from one to the other. We demonstrate that this 385-bp cob fragment is promising for lineage-wise dinoflagellate species identification, given an adequate database.DNA barcoding is a diagnostic technique for species identification using a short, standardized DNA (i.e., DNA barcode) (15). For microbial organisms, this PCR-based technique is useful not only for identifying cultured species but also for rapid retrieval and species identification for uncultured taxa from natural environments. A good DNA barcoding marker should be simple (easy to PCR amplify and sequence) and universal (effective for a wide range of lineages), with a high resolving power (high interspecific and low intraspecific variations). Therefore, an ideal DNA barcoding marker is a relatively short and reasonably variable gene fragment (for species discrimination) flanked by highly conserved sequences (for primer design). The pioneering DNA barcoding work used mitochondrial cytochrome c oxidase 1 (cox1) to identify animal species (9, 10). Mitochondrial genes are a good barcode choice for animals, because they are markedly more variable than nuclear genes (3, 32) and contain conserved regions for primer design. Among other organisms, cox1 has also been shown to be useful for barcoding other organisms, such as fungi (35). Initial attempts at cox1 barcoding for macroalgae (rhodophyte and phaeophyte) also showed good potential (21,29,34). In land plants, the mitochondrial genome evolves substantially more slowly than the nuclear genome (26, 27), rendering its genes less useful than genes...

Spliced leader–based metatranscriptomic analyses lead to recognition of hidden genomic features in dinoflagellates

“…The sequences were aligned with ClustalX, and the alignment was manually inspected and corrected. Phylogenetic analyses were performed according to our previous reported methods (34,35).…”

Recent radiation in a marine and freshwater dinoflagellate species flock

“…LSU rDNA has been successfully used for dinoflagellate species separation (Daugbjerg et al, 2000). The mitochondrial COB gene has been suggested for dinoflagellate barcoding (Lin et al, 2009). ITS2 sequences are highly variable and useful to delineate closely related species of dinoflagellates (Montresor et al, 2003;Litaker et al, 2007).…”

sxtA -Based Quantitative Molecular Assay To Identify Saxitoxin-Producing Harmful Algal Blooms in Marine Waters

“…DNA was extracted from the cell pellets using the cetyltrimethylammonium bromide (CTAB) method (11), with an additional overnight DNA precipitation at Ϫ20°C. Quality and quantity of DNA were determined using a Nanodrop instrument (Thermoscientific) and by amplifying a control dinoflagellate gene (cytb or small-subunit [SSU] rRNA), according to protocols described previously (37), using the primer pair consisting of 4f and 6r, which amplify a 440-bp fragment, or 18S ribosomal DNA (rDNA) primers 18SF08 (5Ј-TTGAT CCTGCCAGTAGTCATATGCTTG-3Ј) and R0ITS (5Ј-CCTTGTTACGACTT CTCCTTCCTC-3Ј), which amplify ϳ1,780 bp (46). sxt qPCR assay development and copy number determination.…”