Molecular characterization of a parasitic tapeworm (Ligula) based on DNA sequences from formalin-fixed specimens

Museum specimens of Ligula (Pseudophyllidea, Ligulidae), a fish parasite tapeworm, that have been preserved in ethanol or fixed permanently in formalin up to 24 years were used for DNA extraction and molecular characterization. DNA was amplified via PCR from samples collected from different fish hosts that lived in both salt and fresh water bodies in the Chinese Qinghai-Tibet Plateau, Russia, and England. Phylogenetic analyses based on partial nucleotide sequences of the 5'-end of nuclear 28S rRNA gene and the mitochondrial cytochrome c oxidase subunit I (COI) gene support the morphologically based taxonomy that groups the Chinese Ligula within the same species as their Europe counterpart: Lingula intestinalis. No nucleotide variation was detected in either the 28S rRNA gene or the COI gene among the seven plerocercoid samples, suggesting a considerable genetic homogeneity among Ligula from different regions. Our results show that geographic isolation, affinity of hosts, and host habitats are not reliable taxonomic criteria for Ligula classification. Our data also indicate a low genetic diversity in the Ligula prevalent in China and in Europe. Our experiments demonstrate that endogenous DNA from specimens that were subjected to permanent formalin fixation can be routinely amplified from parasitic tapeworms, suggesting that fixation time in formalin may not be a critical factor affecting DNA degradation in such museum specimens.