Molecular Phylogenetic Analysis Of Mermithid Nematodes Inferred From 18SrDNA, COI And D₃ Sequences

Mermithidae is a group of nematodes that parasitize on insects with diversified morphological characters and host specificity. Because of their biological control value, it is important to study genetic variations and phylogenetic relations of these entomopatho--genic nematodes. However, the classification and evolutionary relationships of them have always been hindered by the overwhelming number of species, global distribution, and the lack of morphological specializations traditionally used in nematode classification. The confusion and debates have brought much trouble not only to the study of the phyologenetic relationships among groupers, but also to the biological control. The molecular systematic research on Mermithidae is still very limited. It was reported that the genetic relationships among five species belonging to four genera of Mermithidae in China using RAPD by Xu (2005) . The phylogenetic relationships of fifty-seven species within fifty-two genera of nematodes (only four species of Mermithidae) based on 18S rDNA were reported by Blaxter (1998) and Vandergast (2003). The study was the complete report to infer the phylogenetic relationships of Mermithidae based on rDNA and mtDNA, and figured out some confusions of classifying system in Mermithidae.The genomic DNA were estracted from 18 species of Mermithidae. 18S rDNA(750bp) , COI(370bp) and D₃ (300bp) had been acquired by PCR amplification and sequenced using specific three pare primers. The 18S rDNA data were combined with homologous sequences of five species of Mermithidae downloaded from GenBnak and six species of Mermithidae studied by Xu to form the analysis matrix. The base frequency, amino acid frequency, base substitution and genetic distance of DNA sequence had been analyzed by using some bio-software such as ClustalX8.1,MEGA3.1. The results showed there were stronger phylogenetic signal in the data. We adopted 18S rDNA,COI and D₃ gene sequences and their combined data set as molecular marker and constructed the phylogenetic trees using Neighbor-joining (NJ) and Maximum parsimony (MP).1. The average content of A, T, G, C of 18S rDNA gene sequence of twenty four species were25. 8%,26. 3%,26. 5% and 21. 4% respectively, and A+T content was 52.1%, G+C content is 47. 9%,which are similar. The two phylogenetic analyses generated similar affinities, with twenty-four nematodes grouped into two distinct clusters. Romanomermis clustered with Amphimermis first, then clustered with Aranimermis. A second cluster was formed by Gastromermis, Heleidomermis, Strelkovimermis, Agamermis, Hexamermis, Ovomermis, Octomyomermis, Thaumamermis, Mermis. Allomermis and four Mermithidae sp.2. The average content of A, T, G, C of COI gene sequence of eighteen species were 29. 1%, 41. 1%, 18. 1% and 11. 7% respectively, and A+T content was 70. 2%, G+C content is 29. 8%,which showed a strong A+T bias. The phylogenetic trees were similar in general, and the relationship indicated that the eighteen species of Mermithidae were grouped into two distinct clusters. In the phylogenetic trees, five Romanomermis (all parasite in larvae of mosquitoes) clustered together, and then they clustered with Amphimermis, Strelkovimermis, Allomermis. Thaumamermis and Gastromermis into one group. Ovomermis sinensis (Lepidoptera parasites) and Hexamermis agrotis (Lepidoptera parasites) grouped together first, then grouped with Agamermis (Homoptera parasites) and Mermithidae sp.AN-2007, finally with Octomyomermis huazhongensis (parasitic in mosquito larvae). The genetic distances of fourteen mermithid species showed that the relationships of intra-genus were nearer than that of inter-genus.3. The average content of A, T, G, C of D₃ gene sequence of fourteen species were 27. 3%, 23. 2%, 30. 8% and 18. 7% respectively, and A+T content was 50. 5%,G+C content is 49.5%,which are similar. R.sichuanensis is different from other four Romanomermis on D3 gene sequence .In the phylogenetic tree, R.sichuanensis and Amphimermis sp.A-2007 grouped together first, then grouped with other four Romanomermis into one group, and finally with Gastromermis, Allomermis and Strelkovimermis. In the second group, Octomyomermis huazhongensis and Amphimermis sp.B-2007 grouped together first, then grouped with O. sinensis. H. agrotis. Agamermis and Mermithidae sp.AN-2007.18S rDNA,COI and D₃ markers were used to detect the genetic distance among several mermithidae species. The results showed that these three molecular markers were all suitable for study phylogenetic relationships among Mermithidae. In addition, 18S rDNA marker was superior to COI and D₃ marker when analyze the relationships of inter-genus and the latter was suitable for intra-genus.