The identification of Chrysochromulina and Prymnesium species (Haptophyta, Prymnesiophyceae) using fluorescent or chemiluminescent oligonucleotide probes: a means for improving studies on toxic algae

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Abstract
Chrysochromulina and Prymnesium are important bloom-forming organisms in marine and brackish waters, respectively. Both genera include toxic species, which are primarily implicated in fish kills. Previous analyses of small subunit (SSU) rDNA sequences from Chrysochromulina and Prymnesium spp. indicate that Chrysochromulina is paraphyletic. C. polylepis, which produced a spectacular, harmful bloom in 1988, is more closely related to toxic Prymnesium species than to most other Chrysochromulina species based on rDNA sequence comparisons. Signatures were identified in the SSU rRNA gene specific for a clade that comprised primarily toxic taxa (C. polylepis, P. parvum, P. patelliferum and P. calathiferum) and that recognized C. polylepis alone. Oligonucleotide probes complementary to these regions were designed, and their specificity tested using dot-blot hybridization on PCR products of the SSU rRNA gene from 28 strains of Chrysochromulina and Prymnesium. Whole-cell hybridizations were performed with FLUOS- as well as Cy3-labelled probes on cultured species from both genera, and were detected with both epifluorescence microscopy and flow cytometry. The probes afforded easy identification of clonal isolates of C. polylepis and a cluster of closely related species including C. polylepis and Prymnesium spp. The feasibility of using these probes for species identification and studies of population dynamics in the field is discussed.

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