| DNA sequence analysis of the first internal transcribed spacer (ITS1) region of the ribosomal DNA and D1-D3 expansion segments of the 28S gene was conducted to characterize the genetic variation of six amphimictic Hoplolaimus species including, H. magnistylus, H. concaudajuvencus, H. galeatus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, and Hoplolaimus sp. 3, and two closely related parthenogenetic species, H. columbus, and H. seinhorsti. PCR-RFLP and sequence analysis of the ITS1 and 28S PCR products revealed that several haplotypes existed in the same genome of H. columbus, H. magnistylus, H. seinhorsti, H. concaudajuvencus, and Hoplolaimus sp. 1. Phylogenetic analyses using the ITS1, and combined D1-D3 expansion segments produced trees with similar topology; H. columbus and H. seinhorsti grouped in one clade and the other six species ( H. galeatus, H. concaudajuvencus, H. magnistylus, Hoplolaimus sp. 1, Hoplolaimus sp. 2, Hoplolaimus sp. 3) grouped in another clade. Molecular analysis supports morphological schemes for this genus to be divided into two groups based on several phenotypic traits derived from morphological evolution. Hoplolaimus species showed that high genetic divergence in rDNA sequence is combined with low morphological diversity. Based on genetic information, we developed multiplex PCR for H. columbus, H. galeatus, and H. magnistylus and successfully amplified each species from mixed populations. In molecular phylogeny, maximum likelihood and maximum parsimony produced similar topology with two distinct clades; one clade consists of all Hoplolaimus species; the other clade consists of the rest of Hoplolaiminae genera. This result suggests that Hoplolaimus is monophyletic. Another clade consists of Aorolaimus, Helicotylenchus, Rotylenchus , and Scutellonema species. The secondary structures of the D2-D3 expansion segments were predicted based on free energy minimization and comparative sequence analysis. The comparative sequence analysis and energy minimization method yield 9 stems in the D2 and 6 stems in the D3 which show complete or partial compensatory base changes. The predicted secondary model and new sequence alignment based on predicted secondary structures for the D2 and D3 expansion segments will provide useful information to assign positional nucleotide homology and reconstruct more reliable phylogenetic trees. |
