A molecular phylogeny of the British species of Callithamnion based on restriction site data

The marine red macroalgae are the only source of the commercially-important phycocolloids, agar/agarose and carrageenan. These polysaccharides are used widely in the food, pharmaceutical and biotechnology industries [ 11. A persistent problem with certain of the red algae is the difficulty of unequivocally identifying species on the basis of conventional morphological criteria, largely because of phenotypic plasticity. Recently, a variety of molecular techniques have been used so that the genomes may be investigated directly, thus avoiding problems of variable phenotypes. The analysis of restriction fragment length polymorphisms (RFLPs) in plastid DNA has proved particularly useful for identifying species in the Rhodophyta [2], however, the method is limited by the need for relatively large quantities of plant material. Other approaches have been based on the p.c.r. amplification of target regions of the genome and complete sequencing of the amplified fragment 131. Although this is undoubtedly the most thorough approach to the problem, it can be rather time consuming and not particularly suited to the analysis of large numbers of plants. To circumvent these problems we developed a rapid method of assessing 'species' limits in the Grucilurio and Gruciluriopsis (Rhodophyta) based on amplification of 18s-rDNA and analysis of restriction fragment length polymorphisms [4]. In this paper we report on the application of this technique to the phylogenetic analysis of British exposed-shore species of Callithamnion (Rhodophyta). The methodology has been described previously [4]. Samples of Cullithamnion grunulatum, C . hookeri, C . sepositum, C . tetrugonum and C. tetricum were obtained from various exposed shores in the U.K. In addition, the related sheltered shore species C. roseurn was included for comparison. Plants were routinely washed in fresh water at the point of collection and preserved by air-drying or freezing. DNA was extracted from individual plants using a CTAB-based method to remove contaminating polysaccharides [4]. Oligonucleotides (Fig. 1) complementary to conserved sequences at the 5' and 3' termini of eukaryote DNA were chosen as primers for p.c.r. amplification of the 18s-rRNA coding sequence.