A molecular approach for the identification of meiofaunal turbellarians (Platyhelminthes,Turbellaria)

In the absence of reliable morphological characters, or in conjunction with morphology-based identifications, meiofaunal turbellarians may also be identified using the nucleotide sequence of a portion of the large subunit of the ribosomal RNA (26/28S rRNA). A 284 base pair-long region of the 26/28S rRNA has been identified by isolating genomic DNA from ten species of turbellarians belonging to four orders, namely, the Proseriata, Macrostomida, Prolecithophora and Acoela. The proseriates had been collected from localities in Europe and Israel and were preserved in ethanol. The remaining turbellarians were isolated from intertidal sediment samples collected from two sites on the Maine and New Hampshire coast, USA in 1992. Amplification of the genomic DNA was carried out using two primers designed to match the nucleotide sequence of a portion of the 26/28S rDNA gene of the terrestrial nematode,Caenorhabditis elegans (Maupas 1900). This area consists of a highly variable, about 150 base pair-long region, called the D3 expansion segment, followed by a very conserved stretch of sequence. When folded into its secondary structure, the conserved region will form stem structures that correspond to helices 15 to 18 of theC. elegans structural model. The sequence alignment program PILEUP was used to perform a cluster analysis (unweighted pair group method using arthmetic averages, UPGMA) on the sequences. This analysis revealed that the helices allow for the classification of the turbellarians at the level of families and above, whereas if the D3 expansion segment itself was included in the analysis, intrageneric and intraspecific groupings could be established.