Intra- and Interindividual Variation in ITS1 ofParagonimus westermani(Trematoda: Digenea) and Related Species: Implications for Phylogenetic Studies

Abstract

We investigated the utility of the ribosomal first internal transcribed spacer (ITS1) for phylogenetic studies on trematodes of the genusParagonimus.Numerous clones containing ITS1 PCR products were sequenced forP. miyazakii, P. macrorchis,and members of theP. ohiraiandP. westermanispecies complexes. Some additional data were obtained by direct sequencing of PCR products. The ITS1 is composed of three distinct regions: the short 5′ end, followed by a tract of approximately 120 nucleotides which occurs a variable number of times in tandem, and the 3′ region, which lacks repeats and is referred to as the “post-repeat” fragment. Sequences from all three regions can be aligned among the species studied. Our initial hypothesis, that the post-repeat region would be valuable for phylogenetic studies within theP. westermanicomplex, was proved wrong. Intraindividual sequence variation inP. westermaniwas sometimes greater than between individuals of the species complex. In theP. ohiraispecies complex, however, sequence variation within individuals was minimal. Possible reasons for these observations are discussed. We also wished to determine whether the length variants sequenced were the dominant variants present inParagonimusspecies. This was done by probing Southern blots of genomic digests with an ITS1 fragment which lacks repeat sequences. There is generally greater abundance of large variants, with much lower abundance of small variants, such as those sequenced. Differences in ITS1 lengths are attributed largely to differing numbers of repeats, though some exceptions (which are discussed) were found.