Identification and phylogenetic analysis of four Tisbe species (Copepoda, Harpacticoida) using DNA restriction site variation1

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Abstract
Since the analysis of phylogenetic relationship in the taxon Tisbe (Copepoda, Harpacticoida) by using morphological methods often leads to controversial results we have tested a molecular approach as an alternative. The restriction site variation in four species of Tisbe and in Artemia salina which was used as an outgroup was analyzed. Eleven different restriction enzymes were used and six different genes (ribosomal, actin-, histone-, ubiquitin-, α- and β-tubulin genes) were tested using heterologous hybridization probes in search of suitable differences which allow a clear identification of the species and which are useful for a phylogenetic analysis. The best results were obtained with 18S rRNA gene sequences which were amplified in vitro by the polymerase chain reaction technique prior to restriction enzyme analysis. The in vitro amplification solved the problems which were encountered with the other analyses due to the relatively small amounts of highly purified DNA which could be isolated from the cultured animals. Every species can be recognized by a typical restriction fragment pattern. No intraspecific variation could be detected in the conserved gene sequences tested. However, even the highly conserved 18SrDNA sequences exhibit sufficient interspecific variation for a phylogenetic analysis. A phylogenetic tree was inferred from the data with a computer program using the Wagner maximum parsimony method. It is discussed that the presented analysis technique can presumably be scaled down to the level of a single animal.