Neptunomonas phycophila sp. nov. isolated from a culture of Symbiodinium sp., a dinoflagellate symbiont of the sea anemone Aiptasia tagetes.

A Gram-stain-negative, facultatively anaerobic, oxidase- and catalase-positive, rod-shaped bacterium, strain SYM1(T), was isolated from a culture of Symbiodinium sp., an algal symbiont of the sea anemone Aiptasia tagetes collected in Puerto Rico. Growth was observed at 4-40 °C (optimum 30 °C), at pH 5.0-11.0 (optimum pH 8.0) and with 0.5-8 % (optimum 2 %) (w/v) NaCl. Phylogenetic analyses of 16S rRNA gene sequences showed that strain SYM1(T) was a member of the genus Neptunomonas with the type strain of Neptunomonas naphthovorans as the closest phylogenetic relative with a pairwise sequence similarity of 98.15 %. However, DNA-DNA relatedness between strain SYM1(T) and N. naphthovorans CIP 106451(T) was 24 %. Moreover, strain SYM1(T) could be distinguished from its closest relative by several phenotypic characteristics such as NaCl, pH and temperature tolerance, nitrate reduction and utilization of carbon substrates. The major cellular fatty acids were C16 : 0, C18 : 1ω7c and summed feature 3 (comprising C16 : 1ω7c and/or iso-C15 : 0 2-OH). The genomic DNA G+C content of strain SYM1(T) was 45 mol%. Ubiquinone-8 (Q-8) was the only respiratory quinone detected. Based on a polyphasic taxonomic characterization, strain SYM1(T) represents a novel species of the genus Neptunomonas, for which the name Neptunomonas phycophila sp. nov. is proposed. The type strain is SYM1(T) ( = LMG 28329(T) = CECT 8716(T)).

MICROSATELLITE GENOTYPING OF SINGLE CELLS OF THE DINOFLAGELLATE SPECIES LINGULODINIUM POLYEDRUM (DINOPHYCEAE): A NOVEL APPROACH FOR MARINE MICROBIAL POPULATION GENETIC STUDIES(1).

In recent years, two new approaches have been introduced in genetic studies of phytoplankton species. One is the application of highly polymorphic microsatellite markers, which allow detailed population genetic studies; the other is the development of methods that enable the direct genetic characterization of single cells as an alternative to clonal cultures. The aim of this study was to combine these two approaches in a method that would allow microsatellite genotyping of single phytoplankton cells, providing a novel tool for high-resolution population genetic studies. The dinoflagellate species Lingulodinium polyedrum (F. Stein) J. D. Dodge was selected as a model organism to develop this novel approach. The method we describe here is based on several key developments: (i) a simple and efficient DNA extraction method for single cells, (ii) the characterization of microsatellite markers for L. polyedrum, (iii) a protocol for the species identification of single cells through the analysis of partial rRNA gene sequences, and (iv) a two-step multiplex PCR protocol for the simultaneous amplification of microsatellite markers and partial rRNA gene sequences from single cells. Our protocol allowed the amplification of up to six microsatellite loci together with either the complete ITS1-5.8S-ITS2 region or a partial 18S region of the ribosomal gene of L. polyedrum from single motile cells and resting cysts. This article describes and evaluates the developed approach and discusses its significance for population genetic studies of L. polyedrum and other phytoplankton species.