Morphological and molecular characterization of two putative Xiphinema americanum group species, X parasimile and X. simile (Nematoda: Dorylaimida) from Serbia.

Problems associated with the taxonomic status of Xiphinema parasimile and X. simile are reviewed. Their accurate identification is difficult due to their high morphological and morphometrical similarity. A comparative morphological and molecular study of these species was done to define their specific status and differences. Lip region morphology showed less variability in comparison with tail shape in both species, which was more variable in X. simile. Differences in lip region were constant between these species, but specimens with similar tail shape rarely occurred. Both species showed considerable overlap in all morphometrical data and a polytomous key is inadequate for their practical identification. In practice, the most reliable approach for accurate determination of X. parasimile and X. simile is the use of a combination of morphometrics with morphology of lip and vulval regions and tail shape. Total DNA was isolated from individual nematodes of both species and the ITS region of the 18S and 26S rDNA and D1-D2 expansion segments of the 26S rDNA were amplified and cloned. The ITS-RFLP patterns clearly revealed X. simile and X. parasimile to be distinct species. The restriction profiles of the D1-D2 amplicons also discriminated these species. D1-D2 sequences revealed no intrapopulation variability and their comparison showed 89% of similarity to each other. These data show that both ribosomal regions are useful to differentiate X. parasimile and X. simile as two distinct species despite similar morphology and morphometrics.