Species diversity in European Haematococcus pluvialis (Chlorophyceae, Volvocales)

Haematococcus pluvialis has received much interest because it can accumulate large quantities of the pigment astaxanthin. While different isolates of H. pluvialis seem to differ considerably in their physiology, their phylogenetic diversity has not yet been thoroughly studied. We studied the species diversity in a set of temperate European strains of H. pluvialis based on internal transcribed spacer (ITS) rDNA and rbcL molecular phylogenies and determined their congruence with morphology and temperature preferences. The ITS rDNA phylogeny resolved three lineages with the European strains. Three additional lineages were not represented by our European strains. Both statistical parsimony network analysis and the general mixed Yule coalescent recovered these six lineages as different species. An ITS1 structure analysis revealed up to three compensatory base changes (CBCs) between each of the three European lineages, while one CBC was found between lineages in the ITS2 secondary structure. Yet the CBCs were not always present in all strains of each lineage. The rbcL was much less variable and separated only two of the three ITS lineages. We propose an epitype for H. pluvialis and describe the two lineages containing European strains as new species, Haematococcus rubicundus and Haematococcus rubens. Although there is broad morphological overlap, strains of H. pluvialis and H. rubens have, on average, more elongated cells, more noticeable cytoplasmic strands and a more (extreme) pear-shaped protoplast apex than H. rubicundus. Optimal temperatures for growth were similar for the three species and varied between 17 degrees C and 23 degrees C, which is lower than reported earlier from other strains. This study provides a robust phylogenetic framework for further ecophysiological study of Haematococcus