Background

Dinoflagellates are an ecologically important group of protists with important functions as primary producers, coral symbionts and in toxic red tides. Although widely studied, the natural diversity of dinoflagellates is not well known. DNA barcoding has been utilized successfully for many protist groups. We used this approach to systematically sample known "species", as a reference to measure the natural diversity in three marine environments.

Methodology/principal findings

In this study, we assembled a large cytochrome c oxidase 1 (COI) barcode database from 8 public algal culture collections plus 3 private collections worldwide resulting in 336 individual barcodes linked to specific cultures. We demonstrate that COI can identify to the species level in 15 dinoflagellate genera, generally in agreement with existing species names. Exceptions were found in species belonging to genera that were generally already known to be taxonomically challenging, such as Alexandrium or Symbiodinium. Using this barcode database as a baseline for cultured dinoflagellate diversity, we investigated the natural diversity in three diverse marine environments (Northeast Pacific, Northwest Atlantic, and Caribbean), including an evaluation of single-cell barcoding to identify uncultivated groups. From all three environments, the great majority of barcodes were not represented by any known cultured dinoflagellate, and we also observed an explosion in the diversity of genera that previously contained a modest number of known species, belonging to Kareniaceae. In total, 91.5% of non-identical environmental barcodes represent distinct species, but only 51 out of 603 unique environmental barcodes could be linked to cultured species using a conservative cut-off based on distances between cultured species.

Conclusions/significance

COI barcoding was successful in identifying species from 70% of cultured genera. When applied to environmental samples, it revealed a massive amount of natural diversity in dinoflagellates. This highlights the extent to which we underestimate microbial diversity in the environment.

Background

DNA barcoding offers an efficient way to determine species identification and to measure biodiversity. For dinoflagellates, an ancient alveolate group of about 2000 described extant species, DNA barcoding studies have revealed large amounts of unrecognized species diversity, most of which is not represented in culture collections. To date, two mitochondrial gene markers, Cytochrome Oxidase I (COI) and Cytochrome b oxidase (COB), have been used to assess DNA barcoding in dinoflagellates, and both failed to amplify all taxa and suffered from low resolution. Nevertheless, both genes yielded many examples of morphospecies showing cryptic speciation and morphologically distinct named species being genetically similar, highlighting the need for a common marker. For example, a large number of cultured Symbiodinium strains have neither taxonomic identification, nor a common measure of diversity that can be used to compare this genus to other dinoflagellates.

Methodology/principal findings

The purpose of this study was to evaluate the Internal Transcribed Spacer units 1 and 2 (ITS) of the rDNA operon, as a high resolution marker for distinguishing species dinoflagellates in culture. In our study, from 78 different species, the ITS barcode clearly differentiated species from genera and could identify 96% of strains to a known species or sub-genus grouping. 8.3% showed evidence of being cryptic species. A quarter of strains identified had no previous species identification. The greatest levels of hidden biodiversity came from Scrippsiella and the Pfiesteriaceae family, whilst Heterocapsa strains showed a high level of mismatch to their given species name.

Conclusions/significance

The ITS marker was successful in confirming species, revealing hidden diversity in culture collections. This marker, however, may have limited use for environmental barcoding due to paralogues, the potential for unidentifiable chimaeras and priming across taxa. In these cases ITS would serve well in combination with other markers or for specific taxon studies.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Diverse microbial ecosystems underpin life in the sea. Among these microbes are many unicellular eukaryotes that span the diversity of the eukaryotic tree of life. However, genetic tractability has been limited to a few species, which do not represent eukaryotic diversity or environmentally relevant taxa. Here, we report on the development of genetic tools in a range of protists primarily from marine environments. We present evidence for foreign DNA delivery and expression in 13 species never before transformed and for advancement of tools for eight other species, as well as potential reasons for why transformation of yet another 17 species tested was not achieved. Our resource in genetic manipulation will provide insights into the ancestral eukaryotic lifeforms, general eukaryote cell biology, protein diversification and the evolution of cellular pathways.