Study on molecular phylogeny of Schistosoma sinensium based on nuclear ribosomal DNA

Objective To determine the phylogenetic relationships between Schistosoma sinensium and other Schistosomatid species using DNA sequence data. Two segments of the nuclear rDNA repeat, the second internal spacer (ITS2) and large subunit (LSU/12S) were selected for sequencing. \ Methods Adult worms stored in 100% methanol were washed 3 times with 0\^1×TE (pH8\^0) and the genomic DNA was extracted by the GNT\|K method. The target regions were amplified by PCR using specific primers. The PCR products were purified before ligation into the plasmid pT\|adv (Clontech). Recombinant plasmids were amplified in E.coli (strain TOP10), extracted and purified using routine methods and then sequenced using M13 primers (F/R) on a Licor long\|read auto\|sequencer. Sequences of related schistosomes were retrieved from GenBank and aligned with our data in the sequence editor ESEE. Gene trees were constructed in PHYLIP (Version 3\^6 alpha July, 2 000) and MEGA (version 2\^0 beta build 3) using both Maximum Parsimony and Neighbor\|Joining methods. For parsimony analysis, all characters were treated as unordered and with equal weights. At least 3 000 cycles of bootstrapping were carried out. For analysis in MEGA, all gap columns were deleted. Schistosomatium douthitti and Trichobilharzia were used as outgroups. \ Results The ITS2 and LSU sequences of Schistosoma sinensium were obtained. The ITS2 sequence of Trichobilharzia sp. was reported here for the first time. \ Conclusion The phylogenetic trees from these data of nuclear rDNA suggested that S\^sinensium belongs to the Asian schistosome group. And this species might be an ancient member in the Asian clade.