ABSTRACT.    The discrimination among colored species belonging to the stichotrich genus Pseudokeronopsis (Ciliophora, Spirotrichea) is known to be tangled because of the extreme similarity of many morphogenetic and morphological features such as the ciliary pattern that can often be overlapping among different species. Moreover, other characters such as the color, the shape, and the arrangement of cortical granules, which recently some authors suggested to be key-characters in species recognition, are liable to difficult observation and subjective interpretation, generating much confusion in the systematics of this taxon. Indeed, the genus Pseudokeronopsis includes species looking brown, red, orange, yellow, as well as several shades of these colors. To get round this difficulty we performed a characterization of representatives of these organisms based on a multidisciplinary approach, i.e. through the analysis of their morphology under the light microscope (in vivo as well as after Feulgen and protargol staining) and the electron microscope (SEM and TEM), the analysis of the 18S rRNA gene sequence, and the analysis of the produced secondary metabolites carried out by on line Liquid Chromatography/Visible/Mass Spectrometric (LC-DAD-MS) techniques. This approach allowed the identification and the description of two new species, i.e. Pseudokeronopsis sp. strain Kero coming from a coastal pond at the mouth of the Serchio River (Pisa) (salinity 0‰, but rather changeable during the seasons), and Pseudokeronopsis sp. strain Oxsard2, collected in a seawater tidal pool near Cagliari. Both these species were morphologically very similar to other Pseudokeronopsis species already described (P. rubra and P. carnea respectively) but significant differences resulted from both the phylogenetic analysis and the secondary metabolites profiling. Not unexpectedly, the outcome of our approach indicates that Pseudokeronopsis is a complex of species, some of which are likely to be cryptic. Secondary metabolites analysis represents a powerful tool for species identification and discrimination within this group of ciliates.
This work was developed within the framework of the project IPFO Interreg III A Italy-France (Sardegna/Corsica/Toscana) and financially supported by Province of Livorno (Italy) and MIUR (PRIN 2006).