Evaluating the ribosomal internal transcribed spacer (ITS) as a candidate dinoflagellate barcode marker.

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.

Axenic culture of the heterotrophic dinoflagellate Pfiesteria shumwayae in a semi-defined medium.

A semi-defined, biphasic culture medium was developed that supported the axenic growth of three strains of the heterotrophic dinoflagellate Pfiesteria shumwayae. Maximum cell yields and division rates in the semi-defined medium ranged from 0.1 x 10(5) to 4.0 x 10(5) cells/ml and 0.5 to 1.7 divisions/day, respectively, and depended on the concentration of the major components in the medium as well as the P. shumwayae strain. The medium contained high concentrations of certain dissolved and particulate organic compounds, including amino acids and lipids. Pfiesteria shumwayae flagellated cells were attracted to insoluble lipids present in the medium and appeared to feed on the lipid particles, suggesting that phagocytosis may be required for growth in axenic culture. Development of a semi-defined medium represents significant progress toward a completely defined axenic culture medium and subsequent determination of the biochemical requirements of P. shumwayae, needed to advance understanding of the nutritional ecology of this species. Further, this medium provides an economical, simplified method for generating high cell densities of P. shumwayae in axenic culture that will facilitate controlled investigations on the physiology and biochemistry of this heterotrophic dinoflagellate.

AXENIC CULTIVATION OF THE HETEROTROPHIC DINOFLAGELLATE PFIESTERIA SHUMWAYAE AND OBSERVATIONS ON FEEDING BEHAVIOR(1).

Pfiesteria shumwayae Glasgow et J. M. Burkh. [=Pseudopfiesteria shumwayae (Glasgow et J. M. Burkh.) Litaker, Steid., P. L. Mason, Shields et P. A. Tester] is a heterotrophic dinoflagellate commonly found in temperate, estuarine waters. P. shumwayae can feed on other protists, fish, and invertebrates, but research on the biochemical requirements of this species has been restricted by the lack of axenic cultures. An undefined, biphasic culture medium was formulated that supported the axenic growth of two of three strains of P. shumwayae. The medium contained chicken egg yolk as a major component. Successful growth depended on the method used to sterilize the medium, and maximum cell yields (10(4) · mL(-1) ) were similar to those attained in previous research when P. shumwayae was cultured with living fish or microalgae. Additionally, P. shumwayae flagellate cells ingested particles present in the biphasic medium, allowing detailed observations of feeding behavior. This research is an initial step toward a chemically defined axenic culture medium and determination of P. shumwayae metabolic requirements.

Growth of iron(III)-reducing bacteria on clay minerals as the sole electron acceptor and comparison of growth yields on a variety of oxidized iron forms.

Smectite clay minerals are abundant in soils and sediments worldwide and are typically rich in Fe. While recent investigations have shown that the structural Fe(III) bound in clay minerals is reduced by microorganisms, previous studies have not tested growth with clay minerals as the sole electron acceptor. Here we have demonstrated that a pure culture of Shewanella oneidensis strain MR-1 as well as enrichment cultures of Fe(III)-reducing bacteria from rice paddy soil and subsurface sediments are capable of conserving energy for growth with the structural Fe(III) bound in smectite clay as the sole electron acceptor. Pure cultures of S. oneidensis were used for more detailed growth rate and yield experiments on various solid- and soluble-phase electron acceptors [smectite, Fe(III) oxyhydroxide FeOOH, Fe(III) citrate, and oxygen] in the same minimal medium. Growth was assessed as direct cell counts or as an increase in cell carbon (measured as particulate organic carbon). Cell counts showed that similar growth of S. oneidensis (10(8) cells ml(-1)) occurred with smectitic Fe(III) and on other Fe forms [amorphous Fe(III) oxyhydroxide, and Fe citrate] or oxygen as the electron acceptor. In contrast, cell yields of S. oneidensis measured as the increase in cell carbon were similar on all Fe forms tested while yields on oxygen were five times higher, in agreement with thermodynamic predictions. Over a range of particle loadings (0.5 to 4 g liter(-1)), the increase in cell number was highly correlated to the amount of structural Fe in smectite reduced. From phylogenetic analysis of the complete 16S rRNA gene sequences, a predominance of clones retrieved from the clay mineral-reducing enrichment cultures were most closely related to the low-G+C gram-positive members of the Bacteria (Clostridium and Desulfitobacterium) and the delta-Proteobacteria (members of the Geobacteraceae). Results indicate that growth with smectitic Fe(III) is similar in magnitude to that with Fe(III) oxide minerals and is dependent upon the mineral surface area available. Iron(III) bound in clay minerals should be considered an important electron acceptor supporting the growth of bacteria in soils or sedimentary environments.