Molecular taxonomy of Dunaliella (Chlorophyceae), with a special focus on D. salina: ITS2 sequences revisited with an extensive geographical sampling

The Internal Transcribed Spacer 2 (ITS2) of the nuclear rDNA cistron is one of the most frequently used regions for phylogenetic analysis in algae [1-3]. Although its application in deep taxonomic levels was initially limited to comparisons of genera within the same family owing to uncertainties in alignment at higher taxonomic levels, the analysis of its secondary structure has provided key solutions to this problem [4]. Thus, the use of an ITS2 secondary structure improves sequence alignments, resulting in a higher robustness and accuracy of phylogenetic reconstructions [5] and providing help to distinguish species [6]. Furthermore, an automatic approach to analysis is possible [7], as a pipeline consisting of the ITS2 Database (annotation/structure prediction), 4SALE (alignment), ProfDistS (inferring phylogenies) and the CBCAnalyzer (distinguishing species) have recently become available (http://its2.bioapps.biozentrum.uni-wuerzburg.de/?about webcite).In Dunaliella (Chlorophyceae), the use of ITS2 secondary structure for phylogenetic analysis has a long tradition [8-13]. The genus Dunaliella comprises twenty-eight recognized species separated in two subgenera, Pascheria (which contains the freshwater species), and Dunaliella (grouping the marine species); the latter is further subdivided into four sections: Tertiolecta, Dunaliella, Viridis and Peirceinae [11]. The species ascribed to these four sections occur in a wide range of marine habitats such as oceans, brine lakes, salt marshes, salt lagoons and salt water dishes near the sea [14], being Dunaliella salina Teodoresco (section Dunaliella) the most studied one. Dunaliella salina, is the most halotolerant eukaryotic photosynthetic organism known to date [14,15] since it shows a remarkable degree of adaptation to a variety of salt concentrations and it accumulates large amounts of carotenes under extremely stressful conditions such as high salinity, low nitrogen levels, and high solar radiation [14]. Nowadays,