Phyologenetic Relationships Among Several Mermithid Nematode Species Inferred From RAPD And 18S RDNA Sequences

Mermithidae is a group of insect's parasitic nematode with diversified morphological characters and host specificity. Because of the biological control value of these entomopathogenic nematodes, it is necessary to study the genetic variation and phylogenetic evolution of them. It was the first report to infer the phylogenetic relationships of Mermithidae from China and figure out some confusions of classifying system in Mermithidae.In this study, the genetic relationships among five species belonging to four genera of Mermithidae were detected using RAPD. Twelve primers were screened from 47 random primers and amplified 161 bands (from 200bp to 3200 bp). 150 (93.17%) of these bands were polymorphic bands. The number of obtaining segments of individual primer was between 11-16, the average was 13.42. Nei's similarity coefficients and genetic distance were calculated using the RAPDistance and MEGA softwares. The phylogenetic trees were constructed with the methods of UPGMA and NJ. The results from two methods of cluster analysis were similar in general, and the relationship indicated that the five species of Mermithidae could be distinguished with RAPD markers and were grouped into two distinct clusters. In the phylogenetic trees, Romanomermis culicivorax and R. wuchangensis (all parasite in larvae of mosquitoes) clustered together, then they clustered with Agamermis changshaensis (parasite in Homoptera) into one group. Ovomermis sinensis (parasite in Lepidopters) and Amphimermis sp. (parasite in Homoptera) clustered into one group. The genetic distances of five mermithid species showed that the relationships of intra-genus were nearer than that of inter-genus (the genetic distance between R. culicivorax and R. wuchangensis was 0.1789, but the genetic distance between inter-genus were 0.4471-0.5488). All these results suggested that RAPD was suitable for analysis of phylogenetic relationships among Mermithidae.Nucleotide sequences of 18S rDNA (773 base pairs) were compared among 13 samples (8 samples were sequenced in this study and 5 samples were from GenBank). A molecular phylogenetic tree was constructed by using maximum parsimony (MP) and neighbor joining (NJ) methodologies. The sequence data revealed considerable variation in 200 nucleotide sites among the sequencesanalyzed, with transitions outnumbering transversions. All two phylogenetic analysis generated similar affinities, with 13 mermithid species of 7 genera grouped into two distinct clusters. The congeners Romanomermis culicivorax and R. wuchangensis clustered with Aranimermis sp. (Araneae parasites), then clustered with Amphimermis sp. and Mermis nigrescens (Orthoptera parasites). A second cluster was formed by Ovomermis sinensis, Agamermis changshaensis and Octomyomermis sp. (parasitic in of mosquito larvae). The genetic distances of these 13 mermithid samples revealed closer intrageneric distances (0.29% nucleotide dissimilarity) relative to intergeneric comparisons (4.33-14.78%). Furthermore, phylogenetic analysis based on the 18S rDNA of 4 unidentified mermithid samples can be used to resolve relationships among Mermithidae.RAPD and 18S rDNA markers were used to detect the genetic distance among several mermithid species. The results showed that these two molecular markers were all suitable for study phylogenetic relationships among Mermithidae. In addition, RAPD marker was superior to 18S rDNA marker when analyse relationships of intra-genus and the latter was suitable for inter-genus.